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- PDB-8g47: KRAS G12C complex with GDP and AMG 510 imaged on a cryo-EM imagin... -

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Basic information

Entry
Database: PDB / ID: 8g47
TitleKRAS G12C complex with GDP and AMG 510 imaged on a cryo-EM imaging scaffold
Components
  • GTPase KRas
  • RCG-33 - Cryo-EM imaging scaffold subunit B fused to DARPin
KeywordsSIGNALING PROTEIN / CryoEM imaging scaffold / Cancer / GTPase
Function / homology
Function and homology information


forebrain astrocyte development / negative regulation of epithelial cell differentiation / regulation of synaptic transmission, GABAergic / type I pneumocyte differentiation / epithelial tube branching involved in lung morphogenesis / Rac protein signal transduction / skeletal muscle cell differentiation / positive regulation of Rac protein signal transduction / Signaling by RAS GAP mutants / Signaling by RAS GTPase mutants ...forebrain astrocyte development / negative regulation of epithelial cell differentiation / regulation of synaptic transmission, GABAergic / type I pneumocyte differentiation / epithelial tube branching involved in lung morphogenesis / Rac protein signal transduction / skeletal muscle cell differentiation / positive regulation of Rac protein signal transduction / Signaling by RAS GAP mutants / Signaling by RAS GTPase mutants / Activation of RAS in B cells / RAS signaling downstream of NF1 loss-of-function variants / RUNX3 regulates p14-ARF / SOS-mediated signalling / Activated NTRK3 signals through RAS / Activated NTRK2 signals through RAS / SHC1 events in ERBB4 signaling / Signalling to RAS / SHC-related events triggered by IGF1R / Activated NTRK2 signals through FRS2 and FRS3 / glial cell proliferation / Estrogen-stimulated signaling through PRKCZ / SHC-mediated cascade:FGFR3 / MET activates RAS signaling / Signaling by PDGFRA transmembrane, juxtamembrane and kinase domain mutants / Signaling by PDGFRA extracellular domain mutants / SHC-mediated cascade:FGFR2 / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / SHC-mediated cascade:FGFR4 / Signaling by FGFR4 in disease / Erythropoietin activates RAS / SHC-mediated cascade:FGFR1 / protein-membrane adaptor activity / FRS-mediated FGFR3 signaling / Signaling by CSF3 (G-CSF) / Signaling by FLT3 ITD and TKD mutants / positive regulation of glial cell proliferation / FRS-mediated FGFR2 signaling / FRS-mediated FGFR4 signaling / Signaling by FGFR3 in disease / homeostasis of number of cells within a tissue / p38MAPK events / Tie2 Signaling / FRS-mediated FGFR1 signaling / Signaling by FGFR2 in disease / striated muscle cell differentiation / GRB2 events in EGFR signaling / SHC1 events in EGFR signaling / EGFR Transactivation by Gastrin / Signaling by FLT3 fusion proteins / FLT3 Signaling / Signaling by FGFR1 in disease / GRB2 events in ERBB2 signaling / CD209 (DC-SIGN) signaling / Ras activation upon Ca2+ influx through NMDA receptor / NCAM signaling for neurite out-growth / SHC1 events in ERBB2 signaling / Downstream signal transduction / Constitutive Signaling by Overexpressed ERBB2 / Insulin receptor signalling cascade / Signaling by phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants / small monomeric GTPase / VEGFR2 mediated cell proliferation / FCERI mediated MAPK activation / Signaling by ERBB2 TMD/JMD mutants / RAF activation / Constitutive Signaling by EGFRvIII / regulation of long-term neuronal synaptic plasticity / Signaling by high-kinase activity BRAF mutants / Signaling by ERBB2 ECD mutants / MAP2K and MAPK activation / visual learning / Signaling by ERBB2 KD Mutants / Signaling by SCF-KIT / G protein activity / cytoplasmic side of plasma membrane / Regulation of RAS by GAPs / Signaling by CSF1 (M-CSF) in myeloid cells / RAS processing / Negative regulation of MAPK pathway / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / GDP binding / MAPK cascade / Signaling by BRAF and RAF1 fusions / DAP12 signaling / Constitutive Signaling by Ligand-Responsive EGFR Cancer Variants / Ca2+ pathway / gene expression / actin cytoskeleton organization / RAF/MAP kinase cascade / neuron apoptotic process / negative regulation of neuron apoptotic process / Ras protein signal transduction / mitochondrial outer membrane / positive regulation of protein phosphorylation / Golgi membrane / focal adhesion
Similarity search - Function
Small GTPase, Ras-type / small GTPase Ras family profile. / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / AMG 510 (bound form) / GTPase KRas
Similarity search - Component
Biological speciessynthetic construct (others)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.19 Å
AuthorsCastells-Graells, R. / Sawaya, M.R. / Yeates, T.O.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM129854 United States
Department of Energy (DOE, United States)DE-FC02-02ER63421 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Cryo-EM structure determination of small therapeutic protein targets at 3 Å-resolution using a rigid imaging scaffold.
Authors: Roger Castells-Graells / Kyle Meador / Mark A Arbing / Michael R Sawaya / Morgan Gee / Duilio Cascio / Emma Gleave / Judit É Debreczeni / Jason Breed / Karoline Leopold / Ankoor Patel / ...Authors: Roger Castells-Graells / Kyle Meador / Mark A Arbing / Michael R Sawaya / Morgan Gee / Duilio Cascio / Emma Gleave / Judit É Debreczeni / Jason Breed / Karoline Leopold / Ankoor Patel / Dushyant Jahagirdar / Bronwyn Lyons / Sriram Subramaniam / Chris Phillips / Todd O Yeates /
Abstract: Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller ...Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller proteins. Here, we obtain structures of small proteins by binding them to a rigid molecular scaffold based on a designed protein cage, revealing atomic details at resolutions reaching 2.9 Å. We apply this system to the key cancer signaling protein KRAS (19 kDa in size), obtaining four structures of oncogenic mutational variants by cryo-EM. Importantly, a structure for the key G12C mutant bound to an inhibitor drug (AMG510) reveals significant conformational differences compared to prior data in the crystalline state. The findings highlight the promise of cryo-EM scaffolds for advancing the design of drug molecules against small therapeutic protein targets in cancer and other human diseases.
History
DepositionFeb 8, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 27, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RCG-33 - Cryo-EM imaging scaffold subunit B fused to DARPin
B: GTPase KRas
hetero molecules


Theoretical massNumber of molelcules
Total (without water)58,1055
Polymers57,0752
Non-polymers1,0303
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein RCG-33 - Cryo-EM imaging scaffold subunit B fused to DARPin


Mass: 35452.441 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#2: Protein GTPase KRas / K-Ras 2 / Ki-Ras / c-K-ras / c-Ki-ras


Mass: 21622.275 Da / Num. of mol.: 1 / Mutation: G12C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KRAS, KRAS2, RASK2 / Production host: Escherichia coli (E. coli) / References: UniProt: P01116, small monomeric GTPase
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#5: Chemical ChemComp-MOV / AMG 510 (bound form) / 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-4-[(2S)-2-methyl-4-propanoylpiperazin-1-yl]-1-[4-methyl-2-(propan-2-yl)pyridin-3-yl]pyrido[2,3-d]pyrimidin-2(1H)-one


Mass: 562.610 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C30H32F2N6O3
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: KRAS G12C complex with GDP and AMG 510 imaged on a cryo-EM imaging scaffold
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 155000 X / Nominal defocus max: 2250 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20_4459refinement
PHENIX1.20_4459refinement
EM software
IDNameVersionCategory
7Cootmodel fitting
9PHENIX1.20_4459model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.19 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 171436 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 82.31 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0022660
ELECTRON MICROSCOPYf_angle_d0.43473605
ELECTRON MICROSCOPYf_chiral_restr0.0371402
ELECTRON MICROSCOPYf_plane_restr0.0032467
ELECTRON MICROSCOPYf_dihedral_angle_d8.9671009

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