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- PDB-9i5e: A Coiled Coil Module Strategy for High-Resolution Cryo-EM Structu... -

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Basic information

Entry
Database: PDB / ID: 9i5e
TitleA Coiled Coil Module Strategy for High-Resolution Cryo-EM Structures of Small Proteins for Drug Discovery
Components
  • Isoform 2B of GTPase KRas,APH2
  • Nanobody 26
KeywordsSTRUCTURAL PROTEIN / cryoem imaging scaffold / cancer / gtpase / nanobodies / coiled-coil
Function / homology
Function and homology information


response to mineralocorticoid / GMP binding / LRR domain binding / response to isolation stress / response to gravity / myoblast proliferation / cardiac muscle cell proliferation / Signaling by RAS GAP mutants / Signaling by RAS GTPase mutants / Activation of RAS in B cells ...response to mineralocorticoid / GMP binding / LRR domain binding / response to isolation stress / response to gravity / myoblast proliferation / cardiac muscle cell proliferation / 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 / Estrogen-stimulated signaling through PRKCZ / SHC-mediated cascade:FGFR3 / MET activates RAS signaling / positive regulation of Ras protein signal transduction / SHC-mediated cascade:FGFR2 / SHC-mediated cascade:FGFR4 / Signaling by PDGFRA transmembrane, juxtamembrane and kinase domain mutants / Signaling by PDGFRA extracellular domain mutants / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / Erythropoietin activates RAS / SHC-mediated cascade:FGFR1 / Signaling by FGFR4 in disease / Signaling by CSF3 (G-CSF) / FRS-mediated FGFR3 signaling / Signaling by FLT3 ITD and TKD mutants / FRS-mediated FGFR2 signaling / FRS-mediated FGFR4 signaling / p38MAPK events / Signaling by FGFR3 in disease / FRS-mediated FGFR1 signaling / Tie2 Signaling / protein-membrane adaptor activity / Signaling by FGFR2 in disease / Signaling by FLT3 fusion proteins / GRB2 events in EGFR signaling / SHC1 events in EGFR signaling / FLT3 Signaling / Signaling by FGFR1 in disease / EGFR Transactivation by Gastrin / NCAM signaling for neurite out-growth / CD209 (DC-SIGN) signaling / liver development / GRB2 events in ERBB2 signaling / Downstream signal transduction / response to glucocorticoid / Insulin receptor signalling cascade / SHC1 events in ERBB2 signaling / Constitutive Signaling by Overexpressed ERBB2 / Ras activation upon Ca2+ influx through NMDA receptor / Signaling by phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants / VEGFR2 mediated cell proliferation / small monomeric GTPase / FCERI mediated MAPK activation / female pregnancy / Signaling by ERBB2 TMD/JMD mutants / Constitutive Signaling by EGFRvIII / Signaling by SCF-KIT / RAF activation / Signaling by high-kinase activity BRAF mutants / Signaling by ERBB2 ECD mutants / MAP2K and MAPK activation / Signaling by ERBB2 KD Mutants / cytokine-mediated signaling pathway / cytoplasmic side of plasma membrane / Signaling by RAF1 mutants / Signaling by CSF1 (M-CSF) in myeloid cells / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / MAPK cascade / Negative regulation of MAPK pathway / RAS processing / Regulation of RAS by GAPs / Signaling by BRAF and RAF1 fusions / positive regulation of cellular senescence / GDP binding / DAP12 signaling / Constitutive Signaling by Ligand-Responsive EGFR Cancer Variants / RAF/MAP kinase cascade / G protein activity / Ca2+ pathway / Ras protein signal transduction / mitochondrial outer membrane / Golgi membrane / focal adhesion / positive regulation of gene expression / positive regulation of cell population proliferation / GTPase activity / endoplasmic reticulum membrane / GTP binding / protein-containing complex binding
Similarity search - Function
Small GTPase, Ras-type / Small GTPase Ras domain 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 / Chem-M1X / GTPase KRas
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.77 Å
AuthorsSamson, C. / Dossou, I. / Steinmetz, A. / Kumar, A. / Mathieu, M. / Rak, A.
Funding support France, 1items
OrganizationGrant numberCountry
Not funded France
CitationJournal: Sci Rep / Year: 2025
Title: Structural determination of small proteins by cryo-EM using a coiled coil module strategy.
Authors: Samson Camille / Dossou Irène / Steinmetz Anke / Kumar Anand / Mathieu Magali / Rak Alexey /
Abstract: Electron cryo-microscopy (Cryo-EM) has traditionally been used for structural determination of proteins larger than 50 kDa. Recently, various approaches, such as fusion to a scaffold or the use of ...Electron cryo-microscopy (Cryo-EM) has traditionally been used for structural determination of proteins larger than 50 kDa. Recently, various approaches, such as fusion to a scaffold or the use of DARPins-cages, have been developed to extend its application to smaller proteins. In this study, we determined the structure of the small protein target kRasG12C by fusing it to the coiled-coil motif APH, which is targeted by several nanobodies. This method enabled us to achieve a structure with atomic details at a resolution of 3.7 Å. The kRasG12C structure was bound to the inhibitor drug MRTX849 and GDP, both clearly visible in the density map. This method is advantageous due to its ease of setup and applicability to other targets. Additionally, we investigated several other techniques that can be applied to small proteins, regardless of the presence of a terminal helix. These advancements demonstrate the potential of cryo-EM for detailed structural analysis of a wide range of protein targets, extending cryo-EM application for drug discovery.
History
DepositionJan 28, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 12, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Isoform 2B of GTPase KRas,APH2
M: Nanobody 26
N: Nanobody 26
B: Isoform 2B of GTPase KRas,APH2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)76,19610
Polymers74,0494
Non-polymers2,1476
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Isoform 2B of GTPase KRas,APH2 / K-Ras 2 / Ki-Ras / c-K-ras / c-Ki-ras


Mass: 23465.652 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: fusion protein,fusion protein / Source: (gene. exp.) Homo sapiens (human) / Gene: KRAS, KRAS2, RASK2 / Production host: Escherichia coli (E. coli) / References: UniProt: P01116, small monomeric GTPase
#2: Antibody Nanobody 26


Mass: 13558.856 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#3: Chemical ChemComp-M1X / {(2S)-4-[7-(8-chloronaphthalen-1-yl)-2-{[(2S)-1-methylpyrrolidin-2-yl]methoxy}-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-[(2S)-2-fluoropropanoyl]piperazin-2-yl}acetonitrile


Mass: 606.133 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C32H37ClFN7O2
#4: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
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: cryo-EM structure of kRasG12C fused to the APH2 coiled-coil in complex with nanobody Nb26
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 7 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: DARK FIELD / Nominal magnification: 240000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 30 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 4.72 sec. / Electron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 9509

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21.2_5419model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 187753 / Symmetry type: POINT
RefinementHighest resolution: 3.77 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035442
ELECTRON MICROSCOPYf_angle_d0.5647366
ELECTRON MICROSCOPYf_dihedral_angle_d7.108828
ELECTRON MICROSCOPYf_chiral_restr0.04790
ELECTRON MICROSCOPYf_plane_restr0.004940

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