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- EMDB-22191: Yeast TFIIK (Kin28/Ccl1/Tfb3) Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-22191
TitleYeast TFIIK (Kin28/Ccl1/Tfb3) Complex
Map datamain map of TFIIK
Sample
  • Complex: Ternary complex of Kin28-Ccl1-Tfb3 from Saccharomyces cerevisiae.
    • Protein or peptide: RNA polymerase II transcription factor B subunit 3
    • Protein or peptide: Serine/threonine-protein kinase KIN28
    • Protein or peptide: Cyclin CCL1
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ALUMINUM FLUORIDE
Function / homology
Function and homology information


positive regulation of Atg1/ULK1 kinase complex assembly / transcription factor TFIIK complex / transcription factor TFIIH holo complex / cyclin-dependent protein serine/threonine kinase activator activity / [RNA-polymerase]-subunit kinase / cellular response to nitrogen starvation / 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 ...positive regulation of Atg1/ULK1 kinase complex assembly / transcription factor TFIIK complex / transcription factor TFIIH holo complex / cyclin-dependent protein serine/threonine kinase activator activity / [RNA-polymerase]-subunit kinase / cellular response to nitrogen starvation / 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 / RNA polymerase II transcribes snRNA genes / 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 / Formation of TC-NER Pre-Incision Complex / RNA Polymerase I Promoter Escape / Gap-filling DNA repair synthesis and ligation in TC-NER / transcription by RNA polymerase I / Dual incision in TC-NER / 7-methylguanosine mRNA capping / cyclin-dependent protein serine/threonine kinase activity / positive regulation of autophagy / RNA polymerase II CTD heptapeptide repeat kinase activity / nucleotide-excision repair / transcription initiation at RNA polymerase II promoter / positive regulation of transcription elongation by RNA polymerase II / transcription by RNA polymerase II / protein kinase activity / cell division / DNA repair / regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / mitochondrion / ATP binding / nucleus / metal ion binding / 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
Serine/threonine-protein kinase KIN28 / Cyclin CCL1 / RNA polymerase II transcription factor B subunit 3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / Baker's yeast (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.64 Å
Authorsvan Eeuwen T / Murakami K / Li T / Tsai KL
Funding support United States, 1 items
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
DepositionJun 19, 2020-
Header (metadata) releaseApr 28, 2021-
Map releaseApr 28, 2021-
UpdateApr 28, 2021-
Current statusApr 28, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.163
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.163
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xi8
  • Surface level: 0.163
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22191.png

Downloads & links

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Map

FileDownload / File: emd_22191.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map of TFIIK
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 224 pix.
= 185.92 Å		0.83 Å/pix.
x 224 pix.
= 185.92 Å		0.83 Å/pix.
x 224 pix.
= 185.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.163 / Movie #1: 0.163
Minimum - Maximum-0.019227175 - 2.29263
Average (Standard dev.)0.0023950026 (±0.035059486)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 185.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z185.920185.920185.920
α/β/γ90.00090.00090.000
start NX/NY/NZ278280246
NX/NY/NZ474891
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.0192.2930.002

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Supplemental data

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Half map: half-volume 2

Fileemd_22191_half_map_1.map
Annotationhalf-volume 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-volume 2

Fileemd_22191_half_map_2.map
Annotationhalf-volume 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of Kin28-Ccl1-Tfb3 from Saccharomyces cerevisiae.

EntireName: Ternary complex of Kin28-Ccl1-Tfb3 from Saccharomyces cerevisiae.
Components
  • Complex: Ternary complex of Kin28-Ccl1-Tfb3 from Saccharomyces cerevisiae.
    • Protein or peptide: RNA polymerase II transcription factor B subunit 3
    • Protein or peptide: Serine/threonine-protein kinase KIN28
    • Protein or peptide: Cyclin CCL1
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ALUMINUM FLUORIDE

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Supramolecule #1: Ternary complex of Kin28-Ccl1-Tfb3 from Saccharomyces cerevisiae.

SupramoleculeName: Ternary complex of Kin28-Ccl1-Tfb3 from Saccharomyces cerevisiae.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: the yeast Cdk7 complex, that phosphorylates the RNA pol II C-terminal domain (CTD) in transcription initiation.
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightExperimental: 73 KDa

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Macromolecule #1: RNA polymerase II transcription factor B subunit 3

MacromoleculeName: RNA polymerase II transcription factor B subunit 3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Baker's yeast (brewer's yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 7.19103 KDa
SequenceString:
PFNGDREAHP PFTLKGSVYN DPFIKDLEHR KEFIASGFNT NYAYERVLTE AFMGLGCVIS EEL

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Macromolecule #2: Serine/threonine-protein kinase KIN28

MacromoleculeName: Serine/threonine-protein kinase KIN28 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: [RNA-polymerase]-subunit kinase
Source (natural)Organism: Baker's yeast (brewer's yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 34.725043 KDa
SequenceString: VNMEYTKEKK VGEGTYAVVY LGCQHSTGRK IAIKEIKTSE FKDGLDMSAI REVKYLQEMQ HPNVIELIDI FMAYDNLNLV LEFLPTDLE VVIKDKSILF TPADIKAWML MTLRGVYHCH RNFILHRDLK PNNLLFSPDG QIKVADFGLA RAIPAPHEIL (TPO)SNVVTRWY ...String:
VNMEYTKEKK VGEGTYAVVY LGCQHSTGRK IAIKEIKTSE FKDGLDMSAI REVKYLQEMQ HPNVIELIDI FMAYDNLNLV LEFLPTDLE VVIKDKSILF TPADIKAWML MTLRGVYHCH RNFILHRDLK PNNLLFSPDG QIKVADFGLA RAIPAPHEIL (TPO)SNVVTRWY RAPELLFGAK HYTSAIDIWS VGVIFAELML RIPYLPGQND VDQMEVTFRA LGTPTDRDWP EVSSFMT YN KLQIYPPPSR DELRKRFIAA SEYALDFMCG MLTMNPQKRW TAVQCLESDY FKELPPPSDP SSIK

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Macromolecule #3: Cyclin CCL1

MacromoleculeName: Cyclin CCL1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Baker's yeast (brewer's yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 37.552715 KDa
SequenceString: DLYRHSSQYR MWSYTKDQLQ EKRVDTNARA IAYIEENLLK FREAHNLTEE EIKVLEAKAI PLTMEEELDL VNFYAKKVQV IAQHLNLPT EVVATAISFF RRFFLENSVM QIDPKSIVHT TIFLACKSEN YFISVDSFAQ KAKSTRDSVL KFEFKLLESL K FSLLNHHP ...String:
DLYRHSSQYR MWSYTKDQLQ EKRVDTNARA IAYIEENLLK FREAHNLTEE EIKVLEAKAI PLTMEEELDL VNFYAKKVQV IAQHLNLPT EVVATAISFF RRFFLENSVM QIDPKSIVHT TIFLACKSEN YFISVDSFAQ KAKSTRDSVL KFEFKLLESL K FSLLNHHP YKPLHGFFLD IQNVLYGKVD LNYMGQIYDR CKKRITAALL TDVVYFYTPP QITLATLLIE DEALVTRYLE TK FPSREGS QESVPGNEKE EPQNDASTTE KNKEKSTESE EYSIDSAKLL TIIRECKSII EDCKPPSTEE AKKIAAKNYY CQN PSTL

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #5: ALUMINUM FLUORIDE

MacromoleculeName: ALUMINUM FLUORIDE / type: ligand / ID: 5 / Number of copies: 1 / Formula: AF3
Molecular weightTheoretical: 83.977 Da
Chemical component information

ChemComp-AF3:
ALUMINUM FLUORIDE

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.08 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
20.0 mMHEPESHEPES
100.0 mMKoACpotassium acetate
2.5 mMADPadenosine diphosphate
2.0 mMDTTdithiothreitol
2.5 mMAlF3aluminum flouride
GridModel: Quantifoil / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 0 % / Chamber temperature: 293 K / Instrument: LEICA EM CPC
Details: blotted for 2 seconds with Whatman 41 ashless filter paper.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 4620 / Average exposure time: 2.24 sec. / Average electron dose: 45.0 e/Å2
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsMovies collected in super resolution mode and binned during motion correction by MotionCorr2
Particle selectionNumber selected: 3288475
Details: small particle set picked by blob used to generate 2D references used for large scale particle picking
CTF correctionSoftware - Name: CTFFIND (ver. 4.13)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 129955
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final 3D classificationNumber classes: 6 / Avg.num./class: 80000 / Software - Name: RELION (ver. 3.0.8)
Details: combined two most populace classes for final reconstruction.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

1ua2

,

1kxu

)
RefinementProtocol: RIGID BODY FIT
Output model

PDB-6xi8:
Yeast TFIIK (Kin28/Ccl1/Tfb3) Complex

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