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- EMDB-22131: Structure of the human CAK in complex with THZ1 -

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Entry
Database: EMDB / ID: EMD-22131
TitleStructure of the human CAK in complex with THZ1
Map dataPost-processed (sharpened, filtered) cryo-EM map, clipped to 134 pixels (as used for coordinate refinement).
Sample
  • Complex: CDK-activating kinase assembly factor MAT1, Cyclin-H, Cyclin-dependent kinase 7 (E.C.2.7.11.22,2.7.11.23)
    • Protein or peptide: CDK-activating kinase assembly factor MAT1
    • Protein or peptide: Cyclin-H
    • Protein or peptide: Cyclin-dependent kinase 7
  • Ligand: N-(3-{[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino}phenyl)-4-{[4-(dimethylamino)butanoyl]amino}benzamide
KeywordsKinase / transcription / cell cycle / complex / TRANSFERASE
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 / Tat-mediated elongation of the HIV-1 transcript / cyclin-dependent kinase / Formation of HIV-1 elongation complex containing HIV-1 Tat / cyclin-dependent protein serine/threonine kinase activity / Formation of HIV elongation complex in the absence of HIV Tat / Cyclin A/B1/B2 associated events during G2/M transition / Cyclin E associated events during G1/S transition / RNA Polymerase II Transcription Elongation / Cyclin A:Cdk2-associated events at S phase entry / 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 / TP53 Regulates Transcription of DNA Repair Genes / transcription initiation at RNA polymerase II promoter / positive regulation of smooth muscle cell proliferation / RNA Polymerase I Promoter Escape / 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 / response to calcium ion / G1/S transition of mitotic cell cycle / 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 / regulation of cell cycle / protein kinase activity / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGreber BJ / Perez-Bertoldi JM
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM63072 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01-GM063210 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM127018 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: The cryoelectron microscopy structure of the human CDK-activating kinase.
Authors: Basil J Greber / Juan M Perez-Bertoldi / Kif Lim / Anthony T Iavarone / Daniel B Toso / Eva Nogales /
Abstract: The human CDK-activating kinase (CAK), a complex composed of cyclin-dependent kinase (CDK) 7, cyclin H, and MAT1, is a critical regulator of transcription initiation and the cell cycle. It acts by ...The human CDK-activating kinase (CAK), a complex composed of cyclin-dependent kinase (CDK) 7, cyclin H, and MAT1, is a critical regulator of transcription initiation and the cell cycle. It acts by phosphorylating the C-terminal heptapeptide repeat domain of the RNA polymerase II (Pol II) subunit RPB1, which is an important regulatory event in transcription initiation by Pol II, and it phosphorylates the regulatory T-loop of CDKs that control cell cycle progression. Here, we have determined the three-dimensional (3D) structure of the catalytic module of human CAK, revealing the structural basis of its assembly and providing insight into CDK7 activation in this context. The unique third component of the complex, MAT1, substantially extends the interaction interface between CDK7 and cyclin H, explaining its role as a CAK assembly factor, and it forms interactions with the CDK7 T-loop, which may contribute to enhancing CAK activity. We have also determined the structure of the CAK in complex with the covalently bound inhibitor THZ1 in order to provide insight into the binding of inhibitors at the CDK7 active site and to aid in the rational design of therapeutic compounds.
History
DepositionJun 9, 2020-
Header (metadata) releaseSep 9, 2020-
Map releaseSep 9, 2020-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xd3
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22131.png

Downloads & links

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Map

FileDownload / File: emd_22131.map.gz / Format: CCP4 / Size: 9.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed (sharpened, filtered) cryo-EM map, clipped to 134 pixels (as used for coordinate refinement).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.37 Å/pix.
x 134 pix.
= 183.848 Å		1.37 Å/pix.
x 134 pix.
= 183.848 Å		1.37 Å/pix.
x 134 pix.
= 183.848 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.372 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.14176048 - 0.24827321
Average (Standard dev.)0.00016758437 (±0.011323152)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions134134134
Spacing134134134
CellA=B=C: 183.84799 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3721.3721.372
M x/y/z134134134
origin x/y/z0.0000.0000.000
length x/y/z183.848183.848183.848
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ500500500
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS134134134
D min/max/mean-0.1420.2480.000

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Supplemental data

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Mask #1

Fileemd_22131_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unsharpened half-map (full size, 198 pixels).

Fileemd_22131_half_map_1.map
AnnotationUnsharpened half-map (full size, 198 pixels).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unsharpened half-map (full size, 198 pixels).

Fileemd_22131_half_map_2.map
AnnotationUnsharpened half-map (full size, 198 pixels).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CDK-activating kinase assembly factor MAT1, Cyclin-H, Cyclin-depe...

EntireName: CDK-activating kinase assembly factor MAT1, Cyclin-H, Cyclin-dependent kinase 7 (E.C.2.7.11.22,2.7.11.23)
Components
  • Complex: CDK-activating kinase assembly factor MAT1, Cyclin-H, Cyclin-dependent kinase 7 (E.C.2.7.11.22,2.7.11.23)
    • Protein or peptide: CDK-activating kinase assembly factor MAT1
    • Protein or peptide: Cyclin-H
    • Protein or peptide: Cyclin-dependent kinase 7
  • Ligand: N-(3-{[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino}phenyl)-4-{[4-(dimethylamino)butanoyl]amino}benzamide

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Supramolecule #1: CDK-activating kinase assembly factor MAT1, Cyclin-H, Cyclin-depe...

SupramoleculeName: CDK-activating kinase assembly factor MAT1, Cyclin-H, Cyclin-dependent kinase 7 (E.C.2.7.11.22,2.7.11.23)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Recombinantly expressed as a trimeric complex in insect cells (MAT1 subunit truncated) and then modified with covalent inhibitor THZ1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 80 KDa

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Macromolecule #1: CDK-activating kinase assembly factor MAT1

MacromoleculeName: CDK-activating kinase assembly factor MAT1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 10.234531 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
SNAPVTFSTG IKMGQHISLA PIHKLEEALY EYQPLQIETY GPHVPELEML GRLGYLNHVR AASPQDLAGG YTSSLACHRA LQDAFSGLF WQPS

UniProtKB: CDK-activating kinase assembly factor MAT1

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Macromolecule #2: Cyclin-H

MacromoleculeName: Cyclin-H / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.695473 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MYHNSSQKRH WTFSSEEQLA RLRADANRKF RCKAVANGKV LPNDPVFLEP HEEMTLCKYY EKRLLEFCSV FKPAMPRSVV GTACMYFKR FYLNNSVMEY HPRIIMLTCA FLACKVDEFN VSSPQFVGNL RESPLGQEKA LEQILEYELL LIQQLNFHLI V HNPYRPFE ...String:
MYHNSSQKRH WTFSSEEQLA RLRADANRKF RCKAVANGKV LPNDPVFLEP HEEMTLCKYY EKRLLEFCSV FKPAMPRSVV GTACMYFKR FYLNNSVMEY HPRIIMLTCA FLACKVDEFN VSSPQFVGNL RESPLGQEKA LEQILEYELL LIQQLNFHLI V HNPYRPFE GFLIDLKTRY PILENPEILR KTADDFLNRI ALTDAYLLYT PSQIALTAIL SSASRAGITM ESYLSESLML KE NRTCLSQ LLDIMKSMRN LVKKYEPPRS EEVAVLKQKL ERCHSAELAL NVITKKRKGY EDDDYVSKKS KHEEEEWTDD DLV ESL

UniProtKB: Cyclin-H

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Macromolecule #3: Cyclin-dependent kinase 7

MacromoleculeName: Cyclin-dependent kinase 7 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: cyclin-dependent kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.442574 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: SNAMALDVKS RAKRYEKLDF LGEGQFATVY KARDKNTNQI VAIKKIKLGH RSEAKDGINR TALREIKLLQ ELSHPNIIGL LDAFGHKSN ISLVFDFMET DLEVIIKDNS LVLTPSHIKA YMLMTLQGLE YLHQHWILHR DLKPNNLLLD ENGVLKLADF G LAKSFG(SEP)P ...String:
SNAMALDVKS RAKRYEKLDF LGEGQFATVY KARDKNTNQI VAIKKIKLGH RSEAKDGINR TALREIKLLQ ELSHPNIIGL LDAFGHKSN ISLVFDFMET DLEVIIKDNS LVLTPSHIKA YMLMTLQGLE YLHQHWILHR DLKPNNLLLD ENGVLKLADF G LAKSFG(SEP)P NRAYTHQVVT RWYRAPELLF GARMYGVGVD MWAVGCILAE LLLRVPFLPG DSDLDQLTRI FETLGTPT E EQWPDMCSLP DYVTFKSFPG IPLHHIFSAA GDDLLDLIQG LFLFNPCARI TATQALKMKY FSNRPGPTPG CQLPRPNCP VETLKEQSNP ALAIKRKRTE ALEQGGLPKK LIF

UniProtKB: Cyclin-dependent kinase 7

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Macromolecule #4: N-(3-{[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino}phenyl)-4-{...

MacromoleculeName: N-(3-{[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino}phenyl)-4-{[4-(dimethylamino)butanoyl]amino}benzamide
type: ligand / ID: 4 / Number of copies: 1 / Formula: V0G
Molecular weightTheoretical: 568.069 Da
Chemical component information

ChemComp-V0G:
N-(3-{[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino}phenyl)-4-{[4-(dimethylamino)butanoyl]amino}benzamide

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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

Concentration0.2 mg/mL
BufferpH: 7.9
Component:
ConcentrationFormulaName
200.0 mMKCl
5.0 mMMgCl2
20.0 mMHEPES-KOH
5.0 mMbeta-mercaptoethanol
2.0 mMATP-gamma-S
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 5007 / Average exposure time: 2.0 sec. / Average electron dose: 69.0 e/Å2 / Details: 69 frames per movie
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 72886 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.3 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4884787 / Details: 3D-template picking
Startup modelType of model: EMDB MAP
EMDB ID:

22123

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 31198
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)

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Atomic model buiding 1

Initial modelPDB ID:

6xbz


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6xd3:
Structure of the human CAK in complex with THZ1

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