+Open data
-Basic information
Entry | Database: PDB / ID: 8g4e | |||||||||
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Title | Green Fluorescence Protein imaged on a cryo-EM imaging scaffold | |||||||||
Components |
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Keywords | SIGNALING PROTEIN / CryoEM imaging scaffold / Cancer / GTPase | |||||||||
Biological species | synthetic construct (others) Aequorea victoria (jellyfish) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.98 Å | |||||||||
Authors | Castells-Graells, R. / Sawaya, M.R. / Yeates, T.O. | |||||||||
Funding support | United States, 2items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8g4e.cif.gz | 112.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8g4e.ent.gz | 66.8 KB | Display | PDB format |
PDBx/mmJSON format | 8g4e.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8g4e_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 8g4e_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 8g4e_validation.xml.gz | 27.9 KB | Display | |
Data in CIF | 8g4e_validation.cif.gz | 38.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/g4/8g4e ftp://data.pdbj.org/pub/pdb/validation_reports/g4/8g4e | HTTPS FTP |
-Related structure data
Related structure data | 29718MC 8g3kC 8g42C 8g47C 8g4fC 8g4hC M: map data used to model this data C: citing same article (ref.) |
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-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 35287.246 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) |
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#2: Protein | Mass: 26623.918 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Aequorea victoria (jellyfish) / Production host: Escherichia coli (E. coli) |
Has ligand of interest | N |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: sfGFP displayed on a Cryo-EM imaging scaffold / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: synthetic construct (others) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 33 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software |
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1221977 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL / Space: REAL | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 51.16 Å2 | ||||||||||||||||||||||||
Refine LS restraints |
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