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- PDB-6ttq: PKM2 in complex with Compound 10 -

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Basic information

Entry
Database: PDB / ID: 6ttq
TitlePKM2 in complex with Compound 10
ComponentsPyruvate kinase PKM
KeywordsCYTOSOLIC PROTEIN / PKM2 / FBDD
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 / protein tyrosine kinase activity / collagen-containing extracellular matrix / 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 / 1-propan-2-yl-3-pyridin-4-yl-urea / Pyruvate kinase PKM
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 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 30, 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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Structure visualization

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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)241,40512
Polymers239,3274
Non-polymers2,0778
Water50428
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14B
24C
15B
25D
16C
26D

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1111A22 - 531
2111B22 - 531
1121A22 - 531
2121C22 - 531
1131A22 - 531
2131D22 - 531
1141B22 - 531
2141C22 - 531
1151B22 - 531
2151D22 - 531
1161C22 - 531
2161D22 - 531

NCS ensembles :
ID
1
2
3
4
5
6

NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2given(-1, 3.5E-5, -0.000114), (-3.5E-5, -1, 6.3E-5), (-0.000114, 6.3E-5, 1)102.00696, 120.9967, -0.00014
3given(1), (1), (1)
4given(-1, -2.9E-5, 0.000104), (-2.9E-5, 1, -7.0E-5), (-0.000104, -7.0E-5, -1)101.99517, 0.00592, 142.0072
5given(1), (1), (1)
6given(1, 2.8E-5, 0.000104), (2.8E-5, -1, -6.5E-5), (0.000104, 6.5E-5, -1)-0.00782, 121.00264, 141.98845
7given(1), (1), (1)
8given(1, -6.0E-6, 1.0E-5), (-6.0E-6, -1, 7.0E-6), (1.0E-5, -7.0E-6, -1)-8.0E-5, 120.99973, 141.99973
9given(1), (1), (1)
10given(-1, 8.0E-6, 1.0E-5), (8.0E-6, 1, 2.0E-6), (-1.0E-5, 2.0E-6, -1)101.99862, -0.0006, 142.0002
11given(1), (1), (1)
12given(-1, 2.0E-6, 1.0E-6), (-2.0E-6, -1, 6.0E-6), (1.0E-6, 6.0E-6, 1)101.99944, 120.99972, -0.00039

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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: Chemical
ChemComp-NXH / 1-propan-2-yl-3-pyridin-4-yl-urea


Mass: 179.219 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C9H13N3O / Feature type: SUBJECT OF INVESTIGATION
#3: 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
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 28 / 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: 65.4 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 572

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Processing

SoftwareName: REFMAC / Version: 5.8.0222 / Classification: refinement
EM software
IDNameVersionCategory
12RELION3.043D reconstruction
13REFMAC5.8.0222model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 267920
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142864 / Symmetry type: POINT
Atomic model buildingB value: 75.429 / Protocol: OTHER / Space: RECIPROCAL / Target criteria: Maximum likelihood with phases
RefinementCor.coef. Fo:Fc: 0.895 / Highest resolution: 2.7 Å / SU B: 7.972 / SU ML: 0.152 / ESU R: 0.281
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.30097 --
obs0.30097 194751 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 75.429 Å2
Baniso -1Baniso -2Baniso -3
1--0.17 Å20 Å20 Å2
2--1.34 Å20 Å2
3----1.17 Å2
Refinement stepCycle: 1 / Total: 15764
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.010.01515988
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg1.4631.76321608
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.04352036
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.90719.747632
ELECTRON MICROSCOPYr_dihedral_angle_3_deg18.757152588
ELECTRON MICROSCOPYr_dihedral_angle_4_deg16.60415124
ELECTRON MICROSCOPYr_chiral_restr0.1030.22152
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.02111668
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it11.43415.7238156
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it15.36518.9227832
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined25.79995.2820487
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Number: 3901 / Refine-ID: ELECTRON MICROSCOPY / Type: tight thermal / Weight position: 0.87

Ens-IDDom-IDAuth asym-IDRms dev position (Å)
11B0.56
22C0.6
33D0.44
44C0.35
55D0.57
66D0.48
LS refinement shellResolution: 2.7→2.77 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.435 14446 -
obs--100 %

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