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- EMDB-43932: Activated CRAF/MEK heterotetramer from focused refinement of CRAF... -

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Basic information

Entry
Database: EMDB / ID: EMD-43932
TitleActivated CRAF/MEK heterotetramer from focused refinement of CRAF/MEK/14-3-3 complex
Map data
Sample
  • Complex: Activated CRAF/MEK1 heterotetramer complex from focused refinement
    • Protein or peptide: GST26/CRAF chimera
    • Protein or peptide: Dual specificity mitogen-activated protein kinase kinase 1
  • Ligand: N-[3-fluoro-4-({7-[(3-fluoropyridin-2-yl)oxy]-4-methyl-2-oxo-2H-1-benzopyran-3-yl}methyl)pyridin-2-yl]-N'-methylsulfuric diamide
KeywordsInhibitor / complex / SIGNALING PROTEIN / TRANSFERASE
Function / homology
Function and homology information


death-inducing signaling complex assembly / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / intermediate filament cytoskeleton organization / JUN kinase kinase activity / regulation of axon regeneration / mitogen-activated protein kinase kinase / placenta blood vessel development / MAP-kinase scaffold activity / labyrinthine layer development ...death-inducing signaling complex assembly / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / intermediate filament cytoskeleton organization / JUN kinase kinase activity / regulation of axon regeneration / mitogen-activated protein kinase kinase / placenta blood vessel development / MAP-kinase scaffold activity / labyrinthine layer development / cerebellar cortex formation / type B pancreatic cell proliferation / regulation of Rho protein signal transduction / Signaling by MAP2K mutants / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / positive regulation of axonogenesis / insulin secretion involved in cellular response to glucose stimulus / regulation of Golgi inheritance / trachea formation / Negative feedback regulation of MAPK pathway / regulation of early endosome to late endosome transport / regulation of stress-activated MAPK cascade / IFNG signaling activates MAPKs / MAPK3 (ERK1) activation / GP1b-IX-V activation signalling / Frs2-mediated activation / ERBB2-ERBB3 signaling pathway / endodermal cell differentiation / face development / MAP kinase kinase activity / pseudopodium / neurotrophin TRK receptor signaling pathway / Bergmann glial cell differentiation / regulation of cell differentiation / Uptake and function of anthrax toxins / glutathione transferase / thyroid gland development / somatic stem cell population maintenance / protein kinase activator activity / extrinsic apoptotic signaling pathway via death domain receptors / glutathione transferase activity / MAP kinase kinase kinase activity / type II interferon-mediated signaling pathway / positive regulation of protein serine/threonine kinase activity / negative regulation of protein-containing complex assembly / Schwann cell development / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / keratinocyte differentiation / activation of adenylate cyclase activity / positive regulation of peptidyl-serine phosphorylation / response to muscle stretch / myelination / ERK1 and ERK2 cascade / CD209 (DC-SIGN) signaling / glutathione metabolic process / protein serine/threonine/tyrosine kinase activity / insulin-like growth factor receptor signaling pathway / protein serine/threonine kinase activator activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / thymus development / Signal transduction by L1 / cell motility / RAF activation / wound healing / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / Stimuli-sensing channels / neuron differentiation / chemotaxis / Signaling by RAF1 mutants / Negative regulation of MAPK pathway / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / cellular senescence / Signaling by BRAF and RAF1 fusions / MAPK cascade / late endosome / insulin receptor signaling pathway / heart development / protein tyrosine kinase activity / scaffold protein binding / regulation of apoptotic process / mitochondrial outer membrane / eukaryotic translation initiation factor 2alpha kinase activity / early endosome / 3-phosphoinositide-dependent protein kinase activity / DNA-dependent protein kinase activity / ribosomal protein S6 kinase activity / histone H3S10 kinase activity / histone H2AXS139 kinase activity / histone H3S28 kinase activity / histone H4S1 kinase activity / histone H2BS14 kinase activity / histone H3T3 kinase activity / histone H2AS121 kinase activity / Rho-dependent protein serine/threonine kinase activity / histone H2BS36 kinase activity
Similarity search - Function
: / Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / Glutathione S-transferase, C-terminal domain / : / : / Phorbol esters/diacylglycerol binding domain (C1 domain) ...: / Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / Glutathione S-transferase, C-terminal domain / : / : / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. / Zinc finger phorbol-ester/DAG-type profile. / Protein kinase C conserved region 1 (C1) domains (Cysteine-rich domains) / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain / Glutathione S-transferase, N-terminal domain / Glutathione transferase family / Glutathione S-transferase, C-terminal / C1-like domain superfamily / Glutathione S-transferase, C-terminal-like / Soluble glutathione S-transferase C-terminal domain profile. / Soluble glutathione S-transferase N-terminal domain profile. / Glutathione S-transferase, N-terminal / Glutathione S-transferase, C-terminal domain superfamily / Thioredoxin-like superfamily / Ubiquitin-like domain superfamily / 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
RAF proto-oncogene serine/threonine-protein kinase / Glutathione S-transferase class-mu 26 kDa isozyme / Dual specificity mitogen-activated protein kinase kinase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.16 Å
AuthorsQuade B / Cohen SE / Huang X
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cancer Discov / Year: 2024
Title: The Pan-RAF-MEK Nondegrading Molecular Glue NST-628 Is a Potent and Brain-Penetrant Inhibitor of the RAS-MAPK Pathway with Activity across Diverse RAS- and RAF-Driven Cancers.
Authors: Meagan B Ryan / Bradley Quade / Natasha Schenk / Zhong Fang / Marshall Zingg / Steven E Cohen / Brooke M Swalm / Chun Li / Ayşegül Özen / Chaoyang Ye / Maria Stella Ritorto / Xin Huang / ...Authors: Meagan B Ryan / Bradley Quade / Natasha Schenk / Zhong Fang / Marshall Zingg / Steven E Cohen / Brooke M Swalm / Chun Li / Ayşegül Özen / Chaoyang Ye / Maria Stella Ritorto / Xin Huang / Arvin C Dar / Yongxin Han / Klaus P Hoeflich / Michael Hale / Margit Hagel /
Abstract: Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents the ...Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents the phosphorylation and activation of MEK by RAF, overcoming the limitations of traditional RAS-MAPK inhibitors and leading to deep durable inhibition of the pathway. Cellular, biochemical, and structural analyses of RAF-MEK complexes show that NST-628 engages all isoforms of RAF and prevents the formation of BRAF-CRAF heterodimers, a differentiated mechanism from all current RAF inhibitors. With a potent and durable inhibition of the RAF-MEK signaling complex as well as high intrinsic permeability into the brain, NST-628 demonstrates broad efficacy in cellular and patient-derived tumor models harboring diverse MAPK pathway alterations, including orthotopic intracranial models. Given its functional and pharmacokinetic mechanisms that are differentiated from previous therapies, NST-628 is positioned to make an impact clinically in areas of unmet patient need. Significance: This study introduces NST-628, a molecular glue having differentiated mechanism and drug-like properties. NST-628 treatment leads to broad efficacy with high tolerability and central nervous system activity across multiple RAS- and RAF-driven tumor models. NST-628 has the potential to provide transformative clinical benefits as both monotherapy and vertical combination anchor.
History
DepositionMar 6, 2024-
Header (metadata) releaseApr 17, 2024-
Map releaseApr 17, 2024-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43932.png

Downloads & links

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Map

FileDownload / File: emd_43932.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
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AxesZ (Sec.)Y (Row.)X (Col.)
1.81 Å/pix.
x 200 pix.
= 362.1 Å		1.81 Å/pix.
x 200 pix.
= 362.1 Å		1.81 Å/pix.
x 200 pix.
= 362.1 Å

Surface

Projections

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.8105 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0745
Minimum - Maximum-0.1467599 - 0.26997814
Average (Standard dev.)0.000049961604 (±0.008397248)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 362.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43932_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43932_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Activated CRAF/MEK1 heterotetramer complex from focused refinement

EntireName: Activated CRAF/MEK1 heterotetramer complex from focused refinement
Components
  • Complex: Activated CRAF/MEK1 heterotetramer complex from focused refinement
    • Protein or peptide: GST26/CRAF chimera
    • Protein or peptide: Dual specificity mitogen-activated protein kinase kinase 1
  • Ligand: N-[3-fluoro-4-({7-[(3-fluoropyridin-2-yl)oxy]-4-methyl-2-oxo-2H-1-benzopyran-3-yl}methyl)pyridin-2-yl]-N'-methylsulfuric diamide

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Supramolecule #1: Activated CRAF/MEK1 heterotetramer complex from focused refinement

SupramoleculeName: Activated CRAF/MEK1 heterotetramer complex from focused refinement
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: GST26/CRAF chimera

MacromoleculeName: GST26/CRAF chimera / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: glutathione transferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 64.718488 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSPILGYWKI KGLVQPTRLL LEYLEEKYEE HLYERDEGDK WRNKKFELGL EFPNLPYYID GDVKLTQSMA IIRYIADKHN MLGGCPKER AEISMLEGAV LDIRYGVSRI AYSKDFETLK VDFLSKLPEM LKMFEDRLCH KTYLNGDHVT HPDFMLYDAL D VVLYMDPM ...String:
MSPILGYWKI KGLVQPTRLL LEYLEEKYEE HLYERDEGDK WRNKKFELGL EFPNLPYYID GDVKLTQSMA IIRYIADKHN MLGGCPKER AEISMLEGAV LDIRYGVSRI AYSKDFETLK VDFLSKLPEM LKMFEDRLCH KTYLNGDHVT HPDFMLYDAL D VVLYMDPM CLDAFPKLVC FKKRIEAIPQ IDKYLKSSKY IAWPLQGWQA TFGGGDHPPK SDSQPKTPVP AQRERAPVSG TQ EKNKIRP RGQRDSSDDW EIEASEVMLS TRIGSGSFGT VYKGKWHGDV AVKILKVVDP TPEQFQAFRN EVAVLRKTRH VNI LLFMGY MTKDNLAIVT QWCEGSSLYK HLHVQETKFQ MFQLIDIARQ TAQGMDYLHA KNIIHRDMKS NNIFLHEGLT VKIG DFGLA TVKSRWSGSQ QVEQPTGSVL WMAPEVIRMQ DNNPFSFQSD VYSYGIVLYE LMTGELPYSH INNRDQIIFM VGRGY ASPD LSKLYKNCPK AMKRLVADCV KKVKEERPLF PQILSSIELL QHSLPKINRS ASEPSLHRAA HTEDINACTL TTSPRL PVF

UniProtKB: Glutathione S-transferase class-mu 26 kDa isozyme, RAF proto-oncogene serine/threonine-protein kinase

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Macromolecule #2: Dual specificity mitogen-activated protein kinase kinase 1

MacromoleculeName: Dual specificity mitogen-activated protein kinase kinase 1
type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: mitogen-activated protein kinase kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 43.518988 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GMPKKKPTPI QLNPAPDGSA VNGTSSAETN LEALQKKLEE LELDEQQRKR LEAFLTQKQK VGELKDDDFE KISELGAGNG GVVFKVSHK PSGLVMARKL IHLEIKPAIR NQIIRELQVL HECNSPYIVG FYGAFYSDGE ISICMEHMDG GSLDQVLKKA G RIPEQILG ...String:
GMPKKKPTPI QLNPAPDGSA VNGTSSAETN LEALQKKLEE LELDEQQRKR LEAFLTQKQK VGELKDDDFE KISELGAGNG GVVFKVSHK PSGLVMARKL IHLEIKPAIR NQIIRELQVL HECNSPYIVG FYGAFYSDGE ISICMEHMDG GSLDQVLKKA G RIPEQILG KVSIAVIKGL TYLREKHKIM HRDVKPSNIL VNSRGEIKLC DFGVSGQLID AMANAFVGTR SYMSPERLQG TH YSVQSDI WSMGLSLVEM AVGRYPIPPP DAKELELMFG CQVEGDAAET PPRPRTPGRP LSSYGMDSRP PMAIFELLDY IVN EPPPKL PSGVFSLEFQ DFVNKCLIKN PAERADLKQL MVHAFIKRSD AEEVDFAGWL CSTIGLNQPS TPTHAAGV

UniProtKB: Dual specificity mitogen-activated protein kinase kinase 1

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Macromolecule #3: N-[3-fluoro-4-({7-[(3-fluoropyridin-2-yl)oxy]-4-methyl-2-oxo-2H-1...

MacromoleculeName: N-[3-fluoro-4-({7-[(3-fluoropyridin-2-yl)oxy]-4-methyl-2-oxo-2H-1-benzopyran-3-yl}methyl)pyridin-2-yl]-N'-methylsulfuric diamide
type: ligand / ID: 3 / Number of copies: 2 / Formula: A1AHE
Molecular weightTheoretical: 488.464 Da

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

20550

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.16 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 83248
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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