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- EMDB-22123: Structure of the human CDK-activating kinase -

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Basic information

Entry
Database: EMDB / ID: EMD-22123
TitleStructure of the human CDK-activating kinase
Map dataPost-processed map, clipped to 150 pixel box size (as used for coordinate refinement)
Sample
  • Complex: Human CDK-activating kinase
    • Protein or peptide: CDK-activating kinase assembly factor MAT1
    • Protein or peptide: Cyclin-H
    • Protein or peptide: Cyclin-dependent kinase 7
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
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 / ATP-dependent activity, acting on DNA / cyclin-dependent kinase / 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
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: 2.8 Å
AuthorsGreber BJ / Perez-Bertoldi JM / Lim K / Iavarone AT / Toso DB / Nogales E
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)R35-GM127018 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01-GM063210 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 7, 2020-
Header (metadata) releaseSep 9, 2020-
Map releaseSep 9, 2020-
UpdateSep 30, 2020-
Current statusSep 30, 2020Processing 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-6xbz
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22123.png

Downloads & links

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Map

FileDownload / File: emd_22123.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed map, clipped to 150 pixel box size (as used for coordinate refinement)
Voxel sizeX=Y=Z: 1.22 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.15191545 - 0.29542172
Average (Standard dev.)0.00015834851 (±0.0104025155)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 183.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.221.221.22
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z183.000183.000183.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ500500500
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.1520.2950.000

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Supplemental data

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Mask #1

Fileemd_22123_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half-map (full size)

Fileemd_22123_half_map_1.map
AnnotationUnfiltered half-map (full size)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half-map (full size)

Fileemd_22123_half_map_2.map
AnnotationUnfiltered half-map (full size)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human CDK-activating kinase

EntireName: Human CDK-activating kinase
Components
  • Complex: Human CDK-activating kinase
    • Protein or peptide: CDK-activating kinase assembly factor MAT1
    • Protein or peptide: Cyclin-H
    • Protein or peptide: Cyclin-dependent kinase 7
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

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Supramolecule #1: Human CDK-activating kinase

SupramoleculeName: Human CDK-activating kinase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Recombinantly expressed as a trimeric complex in insect cells
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
Molecular weightTheoretical: 100 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: 38.13234 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGSSHHHHHH ENLYFQSNAM DDQGCPRCKT TKYRNPSLKL MVNVCGHTLC ESCVDLLFVR GAGNCPECGT PLRKSNFRVQ LFEDPTVDK EVEIRKKVLK IYNKREEDFP SLREYNDFLE EVEEIVFNLT NNVDLDNTKK KMEIYQKENK DVIQKNKLKL T REQEELEE ...String:
MGSSHHHHHH ENLYFQSNAM DDQGCPRCKT TKYRNPSLKL MVNVCGHTLC ESCVDLLFVR GAGNCPECGT PLRKSNFRVQ LFEDPTVDK EVEIRKKVLK IYNKREEDFP SLREYNDFLE EVEEIVFNLT NNVDLDNTKK KMEIYQKENK DVIQKNKLKL T REQEELEE ALEVERQENE QRRLFIQKEE QLQQILKRKN KQAFLDELES SDLPVALLLA QHKDRSTQLE MQLEKPKPVK PV TFSTGIK MGQHISLAPI HKLEEALYEY QPLQIETYGP HVPELEMLGR LGYLNHVRAA SPQDLAGGYT SSLACHRALQ DAF SGLFWQ PS

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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

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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: 43.731051 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MASWSHPQFE KGGGSGGGSG GGSWSHPQFE KSGGGSENLY FQSNAMALDV KSRAKRYEKL DFLGEGQFAT VYKARDKNTN QIVAIKKIK LGHRSEAKDG INRTALREIK LLQELSHPNI IGLLDAFGHK SNISLVFDFM ETDLEVIIKD NSLVLTPSHI K AYMLMTLQ ...String:
MASWSHPQFE KGGGSGGGSG GGSWSHPQFE KSGGGSENLY FQSNAMALDV KSRAKRYEKL DFLGEGQFAT VYKARDKNTN QIVAIKKIK LGHRSEAKDG INRTALREIK LLQELSHPNI IGLLDAFGHK SNISLVFDFM ETDLEVIIKD NSLVLTPSHI K AYMLMTLQ GLEYLHQHWI LHRDLKPNNL LLDENGVLKL ADFGLAKSFG (SEP)PNRAYTHQV VTRWYRAPEL LFGARMYG V GVDMWAVGCI LAELLLRVPF LPGDSDLDQL TRIFETLGTP TEEQWPDMCS LPDYVTFKSF PGIPLHHIFS AAGDDLLDL IQGLFLFNPC ARITATQALK MKYFSNRPGP TPGCQLPRPN CPVETLKEQS NPALAIKRKR TEALEQGGLP KKLIF

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 1 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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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-mercaptoethanol2-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 - 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
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 FIELDBright-field microscopy / 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
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 (6k x 4k) / #0 - Number grids imaged: 1 / #0 - Number real images: 3296 / #0 - Average exposure time: 2.0 sec. / #0 - Average electron dose: 69.0 e/Å2 / #0 - Details: 69 frames per movie / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 (6k x 4k) / #1 - Number grids imaged: 1 / #1 - Number real images: 4286 / #1 - Average exposure time: 2.0 sec. / #1 - Average electron dose: 69.0 e/Å2 / #1 - Details: 69 frames per movie
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 8982448 / Details: 3D-template picking
CTF correctionSoftware: (Name: RELION (ver. 3.1), CTFFIND (ver. 4))
Details: CTF estimation with CTFFIND4, CTF correction in RELION 3.1 during reconstruction.
Startup modelType of model: INSILICO MODEL
In silico model: Atomic model assembled from PDBs for CDK7 and Cyclin H and converted into 3D-map.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 168906
Image recording ID1

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Atomic model buiding 1

Initial model(PDB ID:

1ua2

,

1kxu

)
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6xbz:
Structure of the human CDK-activating kinase

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