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- EMDB-43221: CryoEM structure of GNE-1952-alkylated KRAS G12C in complex with ... -

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Entry
Database: EMDB / ID: EMD-43221
TitleCryoEM structure of GNE-1952-alkylated KRAS G12C in complex with engineered conformationally rigid Fab 2H11.4DS
Map data
Sample
  • Complex: GNE-1952-alkylated KRAS G12C in complex with Fab 2H11.4DS
    • Complex: GNE-1952-alkylated KRAS G12C
      • Protein or peptide: GTPase KRas
    • Complex: Fab 2H11.4DS
      • Protein or peptide: Fab 2H11.4DS heavy chain
      • Protein or peptide: Fab 2H11.4DS light chain
  • Ligand: 1-{4-[(7M)-6-methyl-7-(5-methyl-2H-indazol-4-yl)quinazolin-4-yl]piperazin-1-yl}propan-1-one
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water
Keywordsfab / antigen binding fragment / protein engineering / gtpase / ONCOPROTEIN-MMUNE SYSTEM complex / ONCOPROTEIN-IMMUNE SYSTEM complex
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 / FRS-mediated FGFR3 signaling / Signaling by CSF3 (G-CSF) / 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 / Insulin receptor signalling cascade / SHC1 events in ERBB2 signaling / Ras activation upon Ca2+ influx through NMDA receptor / 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 / Signaling by ERBB2 TMD/JMD mutants / female pregnancy / RAF activation / 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 / Signaling by ERBB2 KD Mutants / visual learning / 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 speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsKung JE / Sudhamsu J
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: bioRxiv / Year: 2024
Title: Disulfi de constrained Fabs overcome target size limitation for high-resolution single-particle cryo-EM.
Authors: Jennifer E Kung / Matthew C Johnson / Christine C Jao / Christopher P Arthur / Dimitry Tegunov / Alexis Rohou / Jawahar Sudhamsu /
Abstract: High-resolution structures of proteins are critical to understanding molecular mechanisms of biological processes and in the discovery of therapeutic molecules. Cryo-EM has revolutionized structure ...High-resolution structures of proteins are critical to understanding molecular mechanisms of biological processes and in the discovery of therapeutic molecules. Cryo-EM has revolutionized structure determination of large proteins and their complexes, but a vast majority of proteins that underlie human diseases are small (< 50 kDa) and usually beyond its reach due to low signal-to-noise images and difficulties in particle alignment. Current strategies to overcome this problem increase the overall size of small protein targets using scaffold proteins that bind to the target, but are limited by inherent flexibility and not being bound to their targets in a rigid manner, resulting in the target being poorly resolved compared to the scaffolds. Here we present an iteratively engineered molecular design for transforming Fabs (antibody fragments), into conformationally rigid scaffolds (Rigid-Fabs) that, when bound to small proteins (~20 kDa), can enable high-resolution structure determination using cryo-EM. This design introduces multiple disulfide bonds at strategic locations, generates a well-folded Fab constrained into a rigid conformation and can be applied to Fabs from various species, isotypes and chimeric Fabs. We present examples of the Rigid Fab design enabling high-resolution (2.3-2.5 Å) structures of small proteins, Ang2 (26 kDa) and KRAS (21 kDa) by cryo-EM. The strategies for designing disulfide constrained Rigid Fabs in our work thus establish a general approach to overcome the target size limitation of single particle cryo-EM.
History
DepositionDec 27, 2023-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43221.png

Downloads & links

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Map

FileDownload / File: emd_43221.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 224 pix.
= 236.835 Å		1.06 Å/pix.
x 224 pix.
= 236.835 Å		1.06 Å/pix.
x 224 pix.
= 236.835 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0573 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-0.36985233 - 0.7934843
Average (Standard dev.)-0.00035287836 (±0.016096175)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 236.83519 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43221_msk_1.map
Projections & Slices
AxesZYX

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Histogram

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Additional map: Half map for local refinement of KRAS G12C and Fab variable domain

Fileemd_43221_additional_1.map
AnnotationHalf map for local refinement of KRAS G12C and Fab variable domain
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Additional map: Half map for local refinement of KRAS G12C and Fab variable domain

Fileemd_43221_additional_2.map
AnnotationHalf map for local refinement of KRAS G12C and Fab variable domain
Projections & Slices
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Additional map: Local refinement map for KRAS G12C and Fab variable domain

Fileemd_43221_additional_3.map
AnnotationLocal refinement map for KRAS G12C and Fab variable domain
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_43221_half_map_1.map
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Half map: #1

Fileemd_43221_half_map_2.map
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Sample components

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Entire : GNE-1952-alkylated KRAS G12C in complex with Fab 2H11.4DS

EntireName: GNE-1952-alkylated KRAS G12C in complex with Fab 2H11.4DS
Components
  • Complex: GNE-1952-alkylated KRAS G12C in complex with Fab 2H11.4DS
    • Complex: GNE-1952-alkylated KRAS G12C
      • Protein or peptide: GTPase KRas
    • Complex: Fab 2H11.4DS
      • Protein or peptide: Fab 2H11.4DS heavy chain
      • Protein or peptide: Fab 2H11.4DS light chain
  • Ligand: 1-{4-[(7M)-6-methyl-7-(5-methyl-2H-indazol-4-yl)quinazolin-4-yl]piperazin-1-yl}propan-1-one
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: GNE-1952-alkylated KRAS G12C in complex with Fab 2H11.4DS

SupramoleculeName: GNE-1952-alkylated KRAS G12C in complex with Fab 2H11.4DS
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: GNE-1952-alkylated KRAS G12C

SupramoleculeName: GNE-1952-alkylated KRAS G12C / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Fab 2H11.4DS

SupramoleculeName: Fab 2H11.4DS / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: GTPase KRas

MacromoleculeName: GTPase KRas / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 19.352785 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GMTEYKLVVV GACGVGKSAL TIQLIQNHFV DEYDPTIEDS YRKQVVIDGE TSLLDILDTA GQEEYSAMRD QYMRTGEGFL LVFAINNTK SFEDIHHYRE QIKRVKDSED VPMVLVGNKS DLPSRTVDTK QAQDLARSYG IPFIETSAKT RQGVDDAFYT L VREIRKHK EK

UniProtKB: GTPase KRas

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Macromolecule #2: Fab 2H11.4DS heavy chain

MacromoleculeName: Fab 2H11.4DS heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.318248 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EVQLQESGPG CVKPPGTLSL TCAVSGGSIS SSNWWSWVRQ PPGKGLEWIG EIYHSGSTNY NPSLKSRVTI SVDKSKNQFS LKLSSVTAA DTAVYYCARG SSSWYDLGPF DYWGQGTCVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFCECP V TVSWNSGA ...String:
EVQLQESGPG CVKPPGTLSL TCAVSGGSIS SSNWWSWVRQ PPGKGLEWIG EIYHSGSTNY NPSLKSRVTI SVDKSKNQFS LKLSSVTAA DTAVYYCARG SSSWYDLGPF DYWGQGTCVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFCECP V TVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THTHHHHHHP

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Macromolecule #3: Fab 2H11.4DS light chain

MacromoleculeName: Fab 2H11.4DS light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.97749 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: SVLTQPPSAS GTPGQRVTIS CSGSSSNIGS NYVYWYQQLC GTAPKLLIYR NNQRPSGVPD RFSGSKSGTS ASLAISGLRC EDEADYYCA AWDERLSGWV FGGGTKLTVL GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADSSPVK A GVETTTPS ...String:
SVLTQPPSAS GTPGQRVTIS CSGSSSNIGS NYVYWYQQLC GTAPKLLIYR NNQRPSGVPD RFSGSKSGTS ASLAISGLRC EDEADYYCA AWDERLSGWV FGGGTKLTVL GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADSSPVK A GVETTTPS CQSNCNKYAA SSYLSLTPEQ WKSHRSYSCQ VTHEGSTVEK TVAPTECS

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Macromolecule #4: 1-{4-[(7M)-6-methyl-7-(5-methyl-2H-indazol-4-yl)quinazolin-4-yl]p...

MacromoleculeName: 1-{4-[(7M)-6-methyl-7-(5-methyl-2H-indazol-4-yl)quinazolin-4-yl]piperazin-1-yl}propan-1-one
type: ligand / ID: 4 / Number of copies: 1 / Formula: A1AAW
Molecular weightTheoretical: 414.503 Da

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Macromolecule #5: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / 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 #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 40 / 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

BufferpH: 7
Component:
ConcentrationNameFormula
20.0 mMHEPES
100.0 mMsodium chlorideNaCl
5.0 mMmagnesium chlorideMgCl2
5.0 uMGDP
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
Details: The peak fraction was subjected to mild crosslinking with 0.5 mM BS3 at room temperature for 10 min. The crosslinking reaction was quenched by addition of 100mM Tris pH 7.5.
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
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: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 926738
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: cryoSPARC

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Atomic model buiding 1

Initial modelPDB ID:

7rp3


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-8vgq:
CryoEM structure of GNE-1952-alkylated KRAS G12C in complex with engineered conformationally rigid Fab 2H11.4DS

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