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- PDB-8s0r: Cryo-EM structure of CAK modified by covalent inhibitor SY-1365 -

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Basic information

Entry
Database: PDB / ID: 8s0r
TitleCryo-EM structure of CAK modified by covalent inhibitor SY-1365
Components
  • CDK-activating kinase assembly factor MAT1
  • Cyclin-H
  • Cyclin-dependent kinase 7
KeywordsTRANSCRIPTION / Kinase / covalent inhibitor / cell cycle
Function / homology
Function and homology information


ventricular system development / snRNA transcription by RNA polymerase II / CAK-ERCC2 complex / transcription factor TFIIK complex / RNA polymerase II CTD heptapeptide repeat Y1 kinase activity / RNA polymerase II CTD heptapeptide repeat S2 kinase activity / RNA polymerase II CTD heptapeptide repeat T4 kinase activity / RNA polymerase II CTD heptapeptide repeat S5 kinase activity / RNA polymerase II CTD heptapeptide repeat S7 kinase activity / adult heart development ...ventricular system development / snRNA transcription by RNA polymerase II / CAK-ERCC2 complex / transcription factor TFIIK complex / RNA polymerase II CTD heptapeptide repeat Y1 kinase activity / RNA polymerase II CTD heptapeptide repeat S2 kinase activity / RNA polymerase II CTD heptapeptide repeat T4 kinase activity / RNA polymerase II CTD heptapeptide repeat S5 kinase activity / RNA polymerase II CTD heptapeptide repeat S7 kinase activity / adult heart development / transcription factor TFIIH core complex / transcription factor TFIIH holo complex / cyclin-dependent protein serine/threonine kinase activator activity / [RNA-polymerase]-subunit kinase / RNA Polymerase I Transcription Termination / cyclin-dependent protein serine/threonine kinase regulator activity / RNA Pol II CTD phosphorylation and interaction with CE during HIV infection / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / Formation of the HIV-1 Early Elongation Complex / mRNA Capping / HIV Transcription Initiation / RNA Polymerase II HIV Promoter Escape / Transcription of the HIV genome / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / regulation of G1/S transition of mitotic cell cycle / RNA Polymerase I Transcription Initiation / RNA polymerase II transcribes snRNA genes / ATP-dependent activity, acting on DNA / cyclin-dependent kinase / Tat-mediated elongation of the HIV-1 transcript / cyclin-dependent protein serine/threonine kinase activity / Formation of HIV-1 elongation complex containing HIV-1 Tat / Formation of HIV elongation complex in the absence of HIV Tat / Cyclin E associated events during G1/S transition / Cyclin A/B1/B2 associated events during G2/M transition / Cyclin A:Cdk2-associated events at S phase entry / RNA Polymerase II Transcription Elongation / cyclin-dependent protein kinase holoenzyme complex / Formation of RNA Pol II elongation complex / RNA Polymerase II Pre-transcription Events / RNA polymerase II CTD heptapeptide repeat kinase activity / male germ cell nucleus / nucleotide-excision repair / transcription initiation at RNA polymerase II promoter / TP53 Regulates Transcription of DNA Repair Genes / RNA Polymerase I Promoter Escape / positive regulation of smooth muscle cell proliferation / NoRC negatively regulates rRNA expression / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / G1/S transition of mitotic cell cycle / Formation of Incision Complex in GG-NER / fibrillar center / response to calcium ion / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / Cyclin D associated events in G1 / RUNX1 regulates transcription of genes involved in differentiation of HSCs / transcription by RNA polymerase II / protein kinase activity / regulation of cell cycle / protein stabilization / cell division / protein serine kinase activity / DNA repair / protein serine/threonine kinase activity / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / perinuclear region of cytoplasm / positive regulation of transcription by RNA polymerase II / zinc ion binding / nucleoplasm / ATP binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
CyclinH/Ccl1 / Cyclin-dependent kinase 7 / Cdk-activating kinase assembly factor MAT1/Tfb3 / Cdk-activating kinase assembly factor MAT1, centre / CDK-activating kinase assembly factor MAT1 / Zinc finger, C3HC4 type (RING finger) / Cyclin, C-terminal domain 2 / Cyclin C-terminal domain / Cyclin/Cyclin-like subunit Ssn8 / Ubiquitin interacting motif ...CyclinH/Ccl1 / Cyclin-dependent kinase 7 / Cdk-activating kinase assembly factor MAT1/Tfb3 / Cdk-activating kinase assembly factor MAT1, centre / CDK-activating kinase assembly factor MAT1 / Zinc finger, C3HC4 type (RING finger) / Cyclin, C-terminal domain 2 / Cyclin C-terminal domain / Cyclin/Cyclin-like subunit Ssn8 / Ubiquitin interacting motif / Ubiquitin-interacting motif (UIM) domain profile. / Cyclin, N-terminal / Cyclin, N-terminal domain / Cyclin-like / domain present in cyclins, TFIIB and Retinoblastoma / Cyclin-like superfamily / : / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Ring finger / Zinc finger RING-type profile. / Zinc finger, RING-type / Zinc finger, RING/FYVE/PHD-type / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
: / Cyclin-dependent kinase 7 / Cyclin-H / CDK-activating kinase assembly factor MAT1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsFeng, J. / Koh, A.F. / Kotecha, A. / Greber, B.J.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/V009354/1 United Kingdom
CitationJournal: To Be Published
Title: Cryo-EM structure of CAK modified by covalent inhibitor SY-1365
Authors: Jones, T. / Feng, J. / Koh, A.F. / Cronin, N.B. / Kotecha, A. / Marineau, J. / Greber, B.J. / Taatjes, D.
History
DepositionFeb 14, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 25, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: CDK-activating kinase assembly factor MAT1
I: Cyclin-H
J: Cyclin-dependent kinase 7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)87,9084
Polymers87,3193
Non-polymers5891
Water1,892105
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein CDK-activating kinase assembly factor MAT1 / CDK7/cyclin-H assembly factor / Cyclin-G1-interacting protein / Menage a trois / RING finger ...CDK7/cyclin-H assembly factor / Cyclin-G1-interacting protein / Menage a trois / RING finger protein 66 / RING finger protein MAT1 / p35 / p36


Mass: 10234.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MNAT1, CAP35, MAT1, RNF66 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P51948
#2: Protein Cyclin-H / MO15-associated protein / p34 / p37


Mass: 37721.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CCNH / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P51946
#3: Protein Cyclin-dependent kinase 7 / 39 kDa protein kinase / p39 Mo15 / CDK-activating kinase 1 / Cell division protein kinase 7 / ...39 kDa protein kinase / p39 Mo15 / CDK-activating kinase 1 / Cell division protein kinase 7 / Serine/threonine-protein kinase 1 / TFIIH basal transcription factor complex kinase subunit


Mass: 39362.598 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDK7, CAK, CAK1, CDKN7, MO15, STK1 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P50613, cyclin-dependent kinase, [RNA-polymerase]-subunit kinase
#4: Chemical ChemComp-A1H46 / ~{N}-[(1~{S},3~{R})-3-[[5-chloranyl-4-(1~{H}-indol-3-yl)pyrimidin-2-yl]amino]-1-methyl-cyclohexyl]-5-[4-(dimethylamino)butanoylamino]pyridine-2-carboxamide


Mass: 589.131 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H37ClN8O2 / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 105 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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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: CDK-activating kinase / Type: COMPLEX / Details: Human CAK covalently modified by SY-1365 / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.085 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.9
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES-KOH1
2250 mMpotassium chlorideKCl1
32 mMmagnesium chlorideMgCl21
45 mMbeta-mercaptoethanol1
50.7 % (v/v)glycerol1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Tergeo EM plasma cleaner / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 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 FIELD / Calibrated magnification: 245614 X / Nominal defocus max: 1700 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 70 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11975
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv3.3.1particle selection
2EPUimage acquisition
4RELION4.0 betaCTF correction
5cryoSPARCv3.3.1CTF correction
8UCSF ChimeraX1.2.5model fitting
10RELION4.0 betainitial Euler assignment
11RELION4.0 betafinal Euler assignment
12RELION4.0 betaclassification
13RELION4.0 beta3D reconstruction
14PHENIX1.20.1model refinementOPLS4 force field
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 56288 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 8ORM

8orm


Accession code: 8ORM / Source name: PDB / Type: experimental model

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