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- EMDB-17509: Cryo-EM structure of CAK in complex with inhibitor BS-181 -

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Basic information

Entry
Database: EMDB / ID: EMD-17509
TitleCryo-EM structure of CAK in complex with inhibitor BS-181
Map dataPost-processed sharpened cryo-EM map
Sample
  • Complex: 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: ~{N}5-(6-azanylhexyl)-~{N}7-(phenylmethyl)-3-propan-2-yl-pyrazolo[1,5-a]pyrimidine-5,7-diamine
  • Ligand: water
KeywordsKinase / Inhibitor / Transcription / Cell Cycle / TRANSFERASE
Function / homology
Function and homology information


ventricular system development / 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 ...ventricular system development / 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 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 / cyclin-dependent protein serine/threonine kinase activity / Formation of HIV elongation complex in the absence of HIV Tat / ATP-dependent activity, acting on DNA / Cyclin E associated events during G1/S transition / RNA Polymerase II Transcription Elongation / Cyclin A/B1/B2 associated events during G2/M transition / cyclin-dependent protein kinase holoenzyme complex / 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 / nucleotide-excision repair / TP53 Regulates Transcription of DNA Repair Genes / transcription initiation at RNA polymerase II promoter / RNA Polymerase I Promoter Escape / positive regulation of smooth muscle cell proliferation / 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 / G1/S transition of mitotic cell cycle / 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 stabilization / protein kinase activity / 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: 1.9 Å
AuthorsCushing VI / Koh AF / Feng J / Jurgaityte K / Bahl AK / Ali S / Kotecha A / Greber BJ
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/V009354/1 United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: High-resolution cryo-EM of the human CDK-activating kinase for structure-based drug design.
Authors: Victoria I Cushing / Adrian F Koh / Junjie Feng / Kaste Jurgaityte / Alexander Bondke / Sebastian H B Kroll / Marion Barbazanges / Bodo Scheiper / Ash K Bahl / Anthony G M Barrett / Simak ...Authors: Victoria I Cushing / Adrian F Koh / Junjie Feng / Kaste Jurgaityte / Alexander Bondke / Sebastian H B Kroll / Marion Barbazanges / Bodo Scheiper / Ash K Bahl / Anthony G M Barrett / Simak Ali / Abhay Kotecha / Basil J Greber /
Abstract: Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to ...Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to macromolecules refractory to crystallization has been hampered by the often-limiting resolution and throughput of cryogenic electron microscopy (cryo-EM). Here, we use high-resolution cryo-EM to determine structures of the CDK-activating kinase, a master regulator of cell growth and division, in its free and nucleotide-bound states and in complex with 15 inhibitors at up to 1.8 Å resolution. Our structures provide detailed insight into inhibitor interactions and networks of water molecules in the active site of cyclin-dependent kinase 7 and provide insights into the mechanisms contributing to inhibitor selectivity, thereby providing the basis for rational design of next-generation therapeutics. These results establish a methodological framework for the use of high-resolution cryo-EM in structure-based drug design.
History
DepositionMay 30, 2023-
Header (metadata) releaseMar 20, 2024-
Map releaseMar 20, 2024-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17509.png

Downloads & links

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Map

FileDownload / File: emd_17509.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed sharpened cryo-EM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.71 Å/pix.
x 384 pix.
= 273.6 Å		0.71 Å/pix.
x 384 pix.
= 273.6 Å		0.71 Å/pix.
x 384 pix.
= 273.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.7125 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0085
Minimum - Maximum-0.049018476 - 0.080939
Average (Standard dev.)-0.000002656987 (±0.0014732736)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 273.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17509_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

Fileemd_17509_half_map_1.map
AnnotationUnfiltered half-map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half-map

Fileemd_17509_half_map_2.map
AnnotationUnfiltered half-map
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

EntireName: CDK-activating kinase
Components
  • Complex: 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: ~{N}5-(6-azanylhexyl)-~{N}7-(phenylmethyl)-3-propan-2-yl-pyrazolo[1,5-a]pyrimidine-5,7-diamine
  • Ligand: water

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Supramolecule #1: CDK-activating kinase

SupramoleculeName: CDK-activating kinase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 / Details: CAK in complex with non-covalent inhibitor BS-181
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 85 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: 37.721508 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: (ACE)MYHNSSQKR HWTFSSEEQL ARLRADANRK FRCKAVANGK VLPNDPVFLE PHEEMTLCKY YEKRLLEFCS VFKPAM PRS VVGTACMYFK RFYLNNSVME YHPRIIMLTC AFLACKVDEF NVSSPQFVGN LRESPLGQEK ALEQILEYEL LLIQQLN FH LIVHNPYRPF ...String:
(ACE)MYHNSSQKR HWTFSSEEQL ARLRADANRK FRCKAVANGK VLPNDPVFLE PHEEMTLCKY YEKRLLEFCS VFKPAM PRS VVGTACMYFK RFYLNNSVME YHPRIIMLTC AFLACKVDEF NVSSPQFVGN LRESPLGQEK ALEQILEYEL LLIQQLN FH LIVHNPYRPF EGFLIDLKTR YPILENPEIL RKTADDFLNR IALTDAYLLY TPSQIALTAI LSSASRAGIT MESYLSES L MLKENRTCLS QLLDIMKSMR NLVKKYEPPR SEEVAVLKQK LERCHSAELA LNVITKKRKG YEDDDYVSKK SKHEEEEWT DDDLVESL

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: 39.362598 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 LAKSFGSP ...String:
SNAMALDVKS RAKRYEKLDF LGEGQFATVY KARDKNTNQI VAIKKIKLGH RSEAKDGINR TALREIKLLQ ELSHPNIIGL LDAFGHKSN ISLVFDFMET DLEVIIKDNS LVLTPSHIKA YMLMTLQGLE YLHQHWILHR DLKPNNLLLD ENGVLKLADF G LAKSFGSP NRAYTHQVVT RWYRAPELLF GARMYGVGVD MWAVGCILAE LLLRVPFLPG DSDLDQLTRI FETLGTPTEE QW PDMCSLP DYVTFKSFPG IPLHHIFSAA GDDLLDLIQG LFLFNPCARI TATQALKMKY FSNRPGPTPG CQLPRPNCPV ETL KEQSNP ALAIKRKRTE ALEQGGLPKK LIF

UniProtKB: Cyclin-dependent kinase 7

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Macromolecule #4: ~{N}5-(6-azanylhexyl)-~{N}7-(phenylmethyl)-3-propan-2-yl-pyrazolo...

MacromoleculeName: ~{N}5-(6-azanylhexyl)-~{N}7-(phenylmethyl)-3-propan-2-yl-pyrazolo[1,5-a]pyrimidine-5,7-diamine
type: ligand / ID: 4 / Number of copies: 1 / Formula: X2H
Molecular weightTheoretical: 380.53 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 139 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration0.4 mg/mL
BufferpH: 7.9
Component:
ConcentrationFormulaName
200.0 mMKClPotassium chloride
2.0 mMMgCl2Magnesium chloride
20.0 mMHEPES-KOH
5.0 mMBeta-mercaptoethanol
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 50 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Number grids imaged: 1 / Number real images: 5203 / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 245614 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.4 µm
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

Startup modelType of model: EMDB MAP
EMDB ID:

22123

Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 1.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0 beta) / Number images used: 565148
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0 beta)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0 beta)
Final 3D classificationSoftware - Name: RELION (ver. 4.0 beta)

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Atomic model buiding 1

Initial modelPDB ID:

8orm


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8p6w:
Cryo-EM structure of CAK in complex with inhibitor BS-181

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