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- PDB-7kue: CryoEM structure of Yeast TFIIK (Kin28/Ccl1/Tfb3) Complex -

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Basic information

Entry
Database: PDB / ID: 7kue
TitleCryoEM structure of Yeast TFIIK (Kin28/Ccl1/Tfb3) Complex
Components
  • Cyclin CCL1
  • RNA polymerase II transcription factor B subunit 3
  • Serine/threonine-protein kinase KIN28
KeywordsTRANSCRIPTION/Transferase / Polymerase CTD / TFIIH / Phosphorylation / Kinase / CDK / Cyclin / TRANSCRIPTION-Transferase complex
Function / homology
Function and homology information


: / : / : / positive regulation of Atg1/ULK1 kinase complex assembly / : / : / transcription factor TFIIK complex / cellular response to nitrogen starvation / transcription factor TFIIH holo complex / cyclin-dependent protein serine/threonine kinase activator activity ...: / : / : / positive regulation of Atg1/ULK1 kinase complex assembly / : / : / transcription factor TFIIK complex / cellular response to nitrogen starvation / transcription factor TFIIH holo complex / cyclin-dependent protein serine/threonine kinase activator activity / [RNA-polymerase]-subunit kinase / cyclin-dependent protein serine/threonine kinase regulator activity / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / Formation of TC-NER Pre-Incision Complex / RNA polymerase II transcribes snRNA genes / RNA Polymerase I Promoter Escape / TP53 Regulates Transcription of DNA Repair Genes / 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 / RNA Polymerase II Pre-transcription Events / Dual incision in TC-NER / 7-methylguanosine mRNA capping / regulation of cyclin-dependent protein serine/threonine kinase activity / Gap-filling DNA repair synthesis and ligation in TC-NER / transcription by RNA polymerase I / cyclin-dependent protein serine/threonine kinase activity / positive regulation of autophagy / RNA polymerase II CTD heptapeptide repeat kinase activity / transcription initiation at RNA polymerase II promoter / nucleotide-excision repair / transcription by RNA polymerase II / protein kinase activity / cell cycle / cell division / protein phosphorylation / DNA repair / regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / mitochondrion / ATP binding / metal ion binding / nucleus / 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 / Cyclin, N-terminal ...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 / 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. / 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
ADENOSINE-5'-DIPHOSPHATE / ALUMINUM FLUORIDE / Serine/threonine-protein kinase KIN28 / Cyclin CCL1 / RNA polymerase II transcription factor B subunit 3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
Authorsvan Eeuwen, T. / Murakami, K. / Li, T. / Tsai, K.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM123233 United States
CitationJournal: Sci Adv / Year: 2021
Title: Structure of TFIIK for phosphorylation of CTD of RNA polymerase II.
Authors: Trevor van Eeuwen / Tao Li / Hee Jong Kim / Jose J Gorbea Colón / Mitchell I Parker / Roland L Dunbrack / Benjamin A Garcia / Kuang-Lei Tsai / Kenji Murakami /
Abstract: During transcription initiation, the general transcription factor TFIIH marks RNA polymerase II by phosphorylating Ser5 of the carboxyl-terminal domain (CTD) of Rpb1, which is followed by extensive ...During transcription initiation, the general transcription factor TFIIH marks RNA polymerase II by phosphorylating Ser5 of the carboxyl-terminal domain (CTD) of Rpb1, which is followed by extensive modifications coupled to transcription elongation, mRNA processing, and histone dynamics. We have determined a 3.5-Å resolution cryo-electron microscopy (cryo-EM) structure of the TFIIH kinase module (TFIIK in yeast), which is composed of Kin28, Ccl1, and Tfb3, yeast homologs of CDK7, cyclin H, and MAT1, respectively. The carboxyl-terminal region of Tfb3 was lying at the edge of catalytic cleft of Kin28, where a conserved Tfb3 helix served to stabilize the activation loop in its active conformation. By combining the structure of TFIIK with the previous cryo-EM structure of the preinitiation complex, we extend the previously proposed model of the CTD path to the active site of TFIIK.
History
DepositionNov 24, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 28, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
C: RNA polymerase II transcription factor B subunit 3
A: Serine/threonine-protein kinase KIN28
B: Cyclin CCL1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)119,2695
Polymers118,7583
Non-polymers5112
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein RNA polymerase II transcription factor B subunit 3 / RNA polymerase II transcription factor B 38 kDa subunit / RNA polymerase II transcription factor B p38 subunit


Mass: 38128.355 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q03290
#2: Protein Serine/threonine-protein kinase KIN28


Mass: 35369.867 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
References: UniProt: P06242, [RNA-polymerase]-subunit kinase
#3: Protein Cyclin CCL1


Mass: 45259.414 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P37366
#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#5: Chemical ChemComp-AF3 / ALUMINUM FLUORIDE / Aluminium fluoride


Mass: 83.977 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: AlF3
Has ligand of interestN

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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: Ternary complex of Kin28-Ccl1-Tfb3 from Saccharomyces cerevisiae.
Type: COMPLEX
Details: the yeast Cdk7 complex, that phosphorylates the RNA pol II C-terminal domain (CTD) in transcription initiation.
Entity ID: #1-#3 / Source: NATURAL
Molecular weightValue: 0.073 MDa / Experimental value: YES
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: CB010 Tfb3-TAP
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESHEPES1
2100 mMpotassium acetateKoAC1
32.5 mMadenosine diphosphateADPAdenosine diphosphate1
42 mMdithiothreitolDTT1
52.5 mMaluminum flourideAlF31
SpecimenConc.: 0.08 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM CPC / Cryogen name: ETHANE / Humidity: 0 % / Chamber temperature: 293 K
Details: blotted for 2 seconds with Whatman 41 ashless filter paper

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.24 sec. / Electron dose: 45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4620
Details: Images collected in super-resolution mode. Movies were 35 frames. Imaging 1 image/hole, image shift between 4 holes. Focus once per image shift
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

SoftwareName: PHENIX / Version: 1.18.1_3865: / Classification: refinement
EM software
IDNameVersionCategory
2Latitudeimage acquisition
4CTFFIND4.13CTF correction
7UCSF Chimeramodel fitting
9Cootmodel refinement
10PHENIXmodel refinement
11RELION3.0.8initial Euler assignment
12RELION3.0.8final Euler assignment
13RELION3.0.8classification
14RELION3.13D reconstruction
Image processingDetails: Movies collected in super resolution mode and binned during motion correction by MotionCorr2
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3288475
Details: small particle set picked by blob used to generate 2D references used for large scale particle picking
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81466 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building
IDPDB-ID 3D fitting-ID
11UA2

1ua2

1
21KXU

1kxu

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0055104
ELECTRON MICROSCOPYf_angle_d1.4146920
ELECTRON MICROSCOPYf_dihedral_angle_d26.404668
ELECTRON MICROSCOPYf_chiral_restr0.071777
ELECTRON MICROSCOPYf_plane_restr0.01869

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