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- PDB-6tth: PKM2 in complex with L-threonine -

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Basic information

Entry
Database: PDB / ID: 6tth
TitlePKM2 in complex with L-threonine
ComponentsPyruvate kinase PKM
KeywordsCYTOSOLIC PROTEIN / PKM2 / FBDD / L-threonine
Function / homology
Function and homology information


programmed cell death / pyruvate kinase / pyruvate kinase activity / positive regulation of cytoplasmic translation / histone H3T11 kinase activity / canonical glycolysis / Glycolysis / positive regulation of sprouting angiogenesis / potassium ion binding / rough endoplasmic reticulum ...programmed cell death / pyruvate kinase / pyruvate kinase activity / positive regulation of cytoplasmic translation / histone H3T11 kinase activity / canonical glycolysis / Glycolysis / positive regulation of sprouting angiogenesis / potassium ion binding / rough endoplasmic reticulum / glycolytic process / non-specific protein-tyrosine kinase / cilium / cellular response to insulin stimulus / extracellular vesicle / MHC class II protein complex binding / collagen-containing extracellular matrix / protein tyrosine kinase activity / secretory granule lumen / vesicle / ficolin-1-rich granule lumen / transcription coactivator activity / non-specific serine/threonine protein kinase / cadherin binding / phosphorylation / mRNA binding / Neutrophil degranulation / magnesium ion binding / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / mitochondrion / RNA binding / extracellular exosome / extracellular region / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Pyruvate kinase, active site / Pyruvate kinase active site signature. / Pyruvate kinase / Pyruvate kinase, barrel / Pyruvate kinase, insert domain superfamily / Pyruvate kinase, barrel domain / Pyruvate kinase, C-terminal / Pyruvate kinase, C-terminal domain superfamily / Pyruvate kinase, alpha/beta domain / Pyruvate kinase-like, insert domain superfamily ...Pyruvate kinase, active site / Pyruvate kinase active site signature. / Pyruvate kinase / Pyruvate kinase, barrel / Pyruvate kinase, insert domain superfamily / Pyruvate kinase, barrel domain / Pyruvate kinase, C-terminal / Pyruvate kinase, C-terminal domain superfamily / Pyruvate kinase, alpha/beta domain / Pyruvate kinase-like, insert domain superfamily / Pyruvate kinase-like domain superfamily / Pyruvate/Phosphoenolpyruvate kinase-like domain superfamily
Similarity search - Domain/homology
1,6-di-O-phosphono-beta-D-fructofuranose / THREONINE / Pyruvate kinase PKM
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsSaur, M. / Hartshorn, M.J. / Dong, J. / Reeks, J. / Bunkoczi, G. / Jhoti, H. / Williams, P.A.
CitationJournal: Drug Discov Today / Year: 2020
Title: Fragment-based drug discovery using cryo-EM.
Authors: Michael Saur / Michael J Hartshorn / Jing Dong / Judith Reeks / Gabor Bunkoczi / Harren Jhoti / Pamela A Williams /
Abstract: Recent advances in electron cryo-microscopy (cryo-EM) structure determination have pushed the resolutions obtainable by the method into the range widely considered to be of utility for drug discovery. ...Recent advances in electron cryo-microscopy (cryo-EM) structure determination have pushed the resolutions obtainable by the method into the range widely considered to be of utility for drug discovery. Here, we review the use of cryo-EM in fragment-based drug discovery (FBDD) based on in-house method development. We demonstrate not only that cryo-EM can reveal details of the molecular interactions between fragments and a protein, but also that the current reproducibility, quality, and throughput are compatible with FBDD. We exemplify this using the test system β-galactosidase (Bgal) and the oncology target pyruvate kinase 2 (PKM2).
History
DepositionDec 27, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 15, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 25, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.2Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_site / struct_site_gen
Item: _chem_comp.mon_nstd_flag / _chem_comp.name ..._chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Assembly

Deposited unit
A: Pyruvate kinase PKM
B: Pyruvate kinase PKM
C: Pyruvate kinase PKM
D: Pyruvate kinase PKM
hetero molecules


Theoretical massNumber of molelcules
Total (without water)241,16412
Polymers239,3274
Non-polymers1,8378
Water3,459192
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18640 Å2
ΔGint-109 kcal/mol
Surface area58370 Å2
MethodPISA

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Components

#1: Protein
Pyruvate kinase PKM / Cytosolic thyroid hormone-binding protein / CTHBP / Opa-interacting protein 3 / OIP-3 / Pyruvate ...Cytosolic thyroid hormone-binding protein / CTHBP / Opa-interacting protein 3 / OIP-3 / Pyruvate kinase 2/3 / Pyruvate kinase muscle isozyme / Thyroid hormone-binding protein 1 / THBP1 / Tumor M2-PK / p58


Mass: 59831.820 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PKM, OIP3, PK2, PK3, PKM2 / Production host: Escherichia coli (E. coli) / References: UniProt: P14618, pyruvate kinase
#2: Sugar
ChemComp-FBP / 1,6-di-O-phosphono-beta-D-fructofuranose / FRUCTOSE-1,6-BISPHOSPHATE / Fructose 1,6-bisphosphate


Type: D-saccharide, beta linking / Mass: 340.116 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C6H14O12P2
IdentifierTypeProgram
b-D-Fruf1PO36PO3IUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
#3: Chemical
ChemComp-THR / THREONINE / Threonine


Type: L-peptide linking / Mass: 119.119 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C4H9NO3 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 192 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: Pyruvate Kinase M2 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.24 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8.2
SpecimenConc.: 0.16 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 59.98 sec. / Electron dose: 66.8 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 476

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Processing

EM softwareName: RELION / Version: 3.04 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 190577
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 99837 / Symmetry type: POINT

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