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- PDB-7b5q: Cryo-EM structure of the human CAK bound to ICEC0942 (PHENIX-OPLS3e) -

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Basic information

Entry
Database: PDB / ID: 7b5q
TitleCryo-EM structure of the human CAK bound to ICEC0942 (PHENIX-OPLS3e)
Components
  • CDK-activating kinase assembly factor MAT1
  • Cyclin-H
  • Cyclin-dependent kinase 7
KeywordsTRANSCRIPTION / Kinase / protein complex / small molecules inhibitor / CDK-activating kinase
Function / homology
Function and homology information


negative regulation of DNA helicase activity / ventricular system development / cyclin-dependent protein kinase activating kinase holoenzyme complex / snRNA transcription by RNA polymerase II / CAK-ERCC2 complex / transcription factor TFIIK complex / adult heart development / transcription factor TFIIH holo complex / transcription factor TFIIH core complex / cyclin-dependent protein serine/threonine kinase activator activity ...negative regulation of DNA helicase activity / ventricular system development / cyclin-dependent protein kinase activating kinase holoenzyme complex / snRNA transcription by RNA polymerase II / CAK-ERCC2 complex / transcription factor TFIIK complex / adult heart development / transcription factor TFIIH holo complex / transcription factor TFIIH core 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 cyclin-dependent protein serine/threonine kinase activity / regulation of G1/S transition of mitotic cell cycle / RNA Polymerase I Transcription Initiation / RNA polymerase II transcribes snRNA genes / Tat-mediated elongation of the HIV-1 transcript / Formation of HIV-1 elongation complex containing HIV-1 Tat / cyclin-dependent kinase / ATP-dependent activity, acting on DNA / Formation of HIV elongation complex in the absence of HIV Tat / cyclin-dependent protein serine/threonine kinase activity / Cyclin E associated events during G1/S transition / RNA Polymerase II Transcription Elongation / Cyclin A/B1/B2 associated events during G2/M transition / Formation of RNA Pol II elongation complex / Cyclin A:Cdk2-associated events at S phase entry / RNA Polymerase II Pre-transcription Events / RNA polymerase II CTD heptapeptide repeat kinase activity / male germ cell nucleus / transcription initiation at RNA polymerase II promoter / nucleotide-excision repair / RNA Polymerase I Promoter Escape / TP53 Regulates Transcription of DNA Repair Genes / positive regulation of smooth muscle cell proliferation / G1/S transition of mitotic cell cycle / NoRC negatively regulates rRNA expression / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / fibrillar center / Formation of Incision Complex in GG-NER / Dual incision in TC-NER / response to calcium ion / 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 / protein-containing complex assembly / transcription by RNA polymerase II / protein stabilization / regulation of cell cycle / protein kinase activity / cell cycle / cell division / phosphorylation / DNA repair / protein serine kinase activity / 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
Chem-I74 / 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: 2.5 Å
AuthorsGreber, B.J. / Remis, J. / Ali, S. / Nogales, E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM127018 United States
CitationJournal: Biophys J / Year: 2021
Title: 2.5 Å-resolution structure of human CDK-activating kinase bound to the clinical inhibitor ICEC0942.
Authors: Basil J Greber / Jonathan Remis / Simak Ali / Eva Nogales /
Abstract: The human CDK-activating kinase (CAK), composed of CDK7, cyclin H, and MAT1, is involved in the control of transcription initiation and the cell cycle. Because of these activities, it has been ...The human CDK-activating kinase (CAK), composed of CDK7, cyclin H, and MAT1, is involved in the control of transcription initiation and the cell cycle. Because of these activities, it has been identified as a promising target for cancer chemotherapy. A number of CDK7 inhibitors have entered clinical trials, among them ICEC0942 (also known as CT7001). Structural information can aid in improving the affinity and specificity of such drugs or drug candidates, reducing side effects in patients. Here, we have determined the structure of the human CAK in complex with ICEC0942 at 2.5 Å-resolution using cryogenic electron microscopy. Our structure reveals conformational differences of ICEC0942 compared with previous X-ray crystal structures of the CDK2-bound complex, and highlights the critical ability of cryogenic electron microscopy to resolve structures of drug-bound protein complexes without the need to crystalize the protein target.
History
DepositionDec 5, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 10, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 24, 2021Group: Database references / Category: citation / Item: _citation.title
Revision 1.2Mar 3, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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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)119,8734
Polymers119,4793
Non-polymers3951
Water1,31573
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6930 Å2
ΔGint-40 kcal/mol
Surface area26520 Å2
MethodPISA

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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: 38132.340 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: 37695.473 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: 43651.070 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-I74 / (3R,4R)-4-[[[7-[(phenylmethyl)amino]-3-propan-2-yl-pyrazolo[1,5-a]pyrimidin-5-yl]amino]methyl]piperidin-3-ol


Mass: 394.513 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H30N6O / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 73 / 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: CDK-activating kinase bound to ICEC0942 / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.12 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
2200 mMPotassium chlorideKCl1
32 mMMagnesium chlorideMgCl21
45 mMbeta-mercaptoethanol2-Mercaptoethanol1
51 %Glycerol1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Incubated with 50 uM ICEC0942 for 5 min
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 72886 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 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 recording

Imaging-ID: 1 / Average exposure time: 2 sec. / Electron dose: 69 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

IDNum. of grids imagedNum. of real imagesDetails
113571Movies (69 frames) acquired in 3 x 3 pattern by image shift
225302Movies (70 frames) acquired in 3 x 3 pattern by image shift

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.1particle selectiontemplate-based
2SerialEMimage acquisition
4CTFFIND4.1CTF correctionCTF determination
5RELION3.1CTF correctionCTF correction
8UCSF Chimeramodel fitting
9Cootmodel fitting
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
21PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 10904715
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 205478 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Real space refinement in PHENIX combined with the OPLS3e force field (Schrodinger 2020-3).
Atomic model buildingPDB-ID: 6XBZ

6xbz

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