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- EMDB-29720: KRAS G13C complex with GDP imaged on a cryo-EM imaging scaffold -

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Basic information

Entry
Database: EMDB / ID: EMD-29720
TitleKRAS G13C complex with GDP imaged on a cryo-EM imaging scaffold
Map dataKRAS G13C complex with GDP imaged on a cryo-EM imaging scaffold
Sample
  • Complex: KRAS G13C complex with GDP displayed on a Cryo-EM imaging scaffold
    • Protein or peptide: RCG-33 - Cryo-EM imaging scaffold subunit B fused to DARPin
    • Protein or peptide: GTPase KRas
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsCryoEM imaging scaffold / Cancer / GTPase / SIGNALING PROTEIN
Function / homology
Function and homology information


response to mineralocorticoid / GMP binding / forebrain astrocyte development / LRR domain binding / regulation of synaptic transmission, GABAergic / negative regulation of epithelial cell differentiation / response to isolation stress / response to gravity / epithelial tube branching involved in lung morphogenesis / type I pneumocyte differentiation ...response to mineralocorticoid / GMP binding / forebrain astrocyte development / LRR domain binding / regulation of synaptic transmission, GABAergic / negative regulation of epithelial cell differentiation / response to isolation stress / response to gravity / epithelial tube branching involved in lung morphogenesis / type I pneumocyte differentiation / Rac protein signal transduction / positive regulation of Rac protein signal transduction / Signaling by RAS GAP mutants / Signaling by RAS GTPase mutants / Activation of RAS in B cells / myoblast proliferation / skeletal muscle cell differentiation / RAS signaling downstream of NF1 loss-of-function variants / RUNX3 regulates p14-ARF / positive regulation of glial cell proliferation / SOS-mediated signalling / Activated NTRK3 signals through RAS / Activated NTRK2 signals through RAS / SHC1 events in ERBB4 signaling / cardiac muscle cell proliferation / Signalling to RAS / Activated NTRK2 signals through FRS2 and FRS3 / SHC-related events triggered by IGF1R / Estrogen-stimulated signaling through PRKCZ / glial cell proliferation / SHC-mediated cascade:FGFR3 / MET activates RAS signaling / SHC-mediated cascade:FGFR2 / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / Signaling by PDGFRA transmembrane, juxtamembrane and kinase domain mutants / Signaling by PDGFRA extracellular domain mutants / SHC-mediated cascade:FGFR4 / 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 / FRS-mediated FGFR1 signaling / Signaling by FGFR3 in disease / protein-membrane adaptor activity / Tie2 Signaling / striated muscle cell differentiation / Signaling by FGFR2 in disease / GRB2 events in EGFR signaling / SHC1 events in EGFR signaling / Signaling by FLT3 fusion proteins / FLT3 Signaling / Signaling by FGFR1 in disease / EGFR Transactivation by Gastrin / NCAM signaling for neurite out-growth / homeostasis of number of cells within a tissue / CD209 (DC-SIGN) signaling / GRB2 events in ERBB2 signaling / Downstream signal transduction / Ras activation upon Ca2+ influx through NMDA receptor / Insulin receptor signalling cascade / SHC1 events in ERBB2 signaling / response to glucocorticoid / Constitutive Signaling by Overexpressed ERBB2 / Signaling by phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants / VEGFR2 mediated cell proliferation / small monomeric GTPase / FCERI mediated MAPK activation / liver development / RAF activation / Signaling by ERBB2 TMD/JMD mutants / female pregnancy / Signaling by SCF-KIT / Signaling by high-kinase activity BRAF mutants / Constitutive Signaling by EGFRvIII / regulation of long-term neuronal synaptic plasticity / MAP2K and MAPK activation / Signaling by ERBB2 ECD mutants / visual learning / Signaling by ERBB2 KD Mutants / cytoplasmic side of plasma membrane / cytokine-mediated signaling pathway / 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 / Signaling by BRAF and RAF1 fusions / positive regulation of cellular senescence / DAP12 signaling / MAPK cascade / Constitutive Signaling by Ligand-Responsive EGFR Cancer Variants
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
Biological speciessynthetic construct (others) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsCastells-Graells R / Sawaya MR / Yeates TO
Funding support United States, 2 items
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 9, 2023-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateSep 27, 2023-
Current statusSep 27, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29720.png

Downloads & links

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Map

FileDownload / File: emd_29720.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKRAS G13C complex with GDP imaged on a cryo-EM imaging scaffold
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 288 pix.
= 316.8 Å		1.1 Å/pix.
x 288 pix.
= 316.8 Å		1.1 Å/pix.
x 288 pix.
= 316.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-7.377754 - 8.964314999999999
Average (Standard dev.)-0.00056958094 (±0.061784923)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 316.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_29720_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_29720_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : KRAS G13C complex with GDP displayed on a Cryo-EM imaging scaffold

EntireName: KRAS G13C complex with GDP displayed on a Cryo-EM imaging scaffold
Components
  • Complex: KRAS G13C complex with GDP displayed on a Cryo-EM imaging scaffold
    • Protein or peptide: RCG-33 - Cryo-EM imaging scaffold subunit B fused to DARPin
    • Protein or peptide: GTPase KRas
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: KRAS G13C complex with GDP displayed on a Cryo-EM imaging scaffold

SupramoleculeName: KRAS G13C complex with GDP displayed on a Cryo-EM imaging scaffold
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: synthetic construct (others)

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

MacromoleculeName: RCG-33 - Cryo-EM imaging scaffold subunit B fused to DARPin
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 35.452441 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MFTRRGDQGE TDLANRARVG KDSPVVEVQG TIDELNSFIG YALVLSRWDD IRNDLFRIQN DLFVLGEDVS TGGKGRTVTM DMIIYLIKR SVEMKAEIGK IELFVVPGGS VESASLHMAR AVSRRLERRI KAASELTEIN ANVLLYANML SNILFMHALI S NKRKEELD ...String:
MFTRRGDQGE TDLANRARVG KDSPVVEVQG TIDELNSFIG YALVLSRWDD IRNDLFRIQN DLFVLGEDVS TGGKGRTVTM DMIIYLIKR SVEMKAEIGK IELFVVPGGS VESASLHMAR AVSRRLERRI KAASELTEIN ANVLLYANML SNILFMHALI S NKRKEELD KKLLEAARAG QDDEVAALLA KGADVNAHDT FGFTPLHLAA LYGHLEIVEV LLKRGADINA DDSYGRTPLH LA AMRGHLE IVELLLRWGA DVNAADEEGR TPLHLAAKRG HLEIVEVLLK NGADVNAQDK FGKTAFDISI DNGNEDLAEI LQK L

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Macromolecule #2: GTPase KRas

MacromoleculeName: GTPase KRas / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: small monomeric GTPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 19.439863 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSMTEYKLVV VGAGCVGKSA LTIQLIQNHF VDEYDPTIED SYRKQVVIDG ETSLLDILDT AGQEEYSAMR DQYMRTGEGF LLVFAINNT KSFEDIHHYR EQIKRVKDSE DVPMVLVGNK SDLPSRTVDT KQAQDLARSY GIPFIETSAK TRQGVDDAFY T LVREIRKH KEK

UniProtKB: GTPase KRas

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Macromolecule #3: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1653856
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8g4h:
KRAS G13C complex with GDP imaged on a cryo-EM imaging scaffold

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