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Yorodumi- EMDB-29718: Green Fluorescence Protein imaged on a cryo-EM imaging scaffold -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-29718 | |||||||||
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Title | Green Fluorescence Protein imaged on a cryo-EM imaging scaffold | |||||||||
Map data | Green Fluorescence Protein imaged on a cryo-EM imaging scaffold | |||||||||
Sample |
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Keywords | CryoEM imaging scaffold / Cancer / GTPase / SIGNALING PROTEIN | |||||||||
Biological species | synthetic construct (others) / Aequorea victoria (jellyfish) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.98 Å | |||||||||
Authors | Castells-Graells R / Sawaya MR / Yeates TO | |||||||||
Funding support | United States, 2 items
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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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_29718.map.gz | 59.6 MB | EMDB map data format | |
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Header (meta data) | emd-29718-v30.xml emd-29718.xml | 16.6 KB 16.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_29718_fsc.xml | 6.9 KB | Display | FSC data file |
Images | emd_29718.png | 47.5 KB | ||
Filedesc metadata | emd-29718.cif.gz | 6 KB | ||
Others | emd_29718_half_map_1.map.gz emd_29718_half_map_2.map.gz | 59.3 MB 59.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-29718 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-29718 | HTTPS FTP |
-Validation report
Summary document | emd_29718_validation.pdf.gz | 808 KB | Display | EMDB validaton report |
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Full document | emd_29718_full_validation.pdf.gz | 807.5 KB | Display | |
Data in XML | emd_29718_validation.xml.gz | 16 KB | Display | |
Data in CIF | emd_29718_validation.cif.gz | 20.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-29718 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-29718 | HTTPS FTP |
-Related structure data
Related structure data | 8g4eMC 8g3kC 8g42C 8g47C 8g4fC 8g4hC M: atomic model generated by this map C: citing same article (ref.) |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_29718.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Green Fluorescence Protein imaged on a cryo-EM imaging scaffold | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.2375 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: Half Map 1
File | emd_29718_half_map_1.map | ||||||||||||
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Annotation | Half Map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half Map 2
File | emd_29718_half_map_2.map | ||||||||||||
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Annotation | Half Map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : sfGFP displayed on a Cryo-EM imaging scaffold
Entire | Name: sfGFP displayed on a Cryo-EM imaging scaffold |
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Components |
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-Supramolecule #1: sfGFP displayed on a Cryo-EM imaging scaffold
Supramolecule | Name: sfGFP displayed on a Cryo-EM imaging scaffold / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: synthetic construct (others) |
-Macromolecule #1: RCG-10 - Cryo-EM imaging scaffold subunit B fused to DARPin
Macromolecule | Name: RCG-10 - Cryo-EM imaging scaffold subunit B fused to DARPin type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 35.287246 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 YDDQVAALLA KGADVNAADD VGVTPLHLAA QRGHLEIVEV LLKRGADINA ADLWGQTPLH LA ATAGHLE IVELLLRWGA DVNARDNIGH TPLHLAAWAG HLEIVEVLLK YGADVNAQDK FGKTPFDLAI DNGNEDIAEV LQK AA |
-Macromolecule #2: Superfolder Green Fluorescent Protein
Macromolecule | Name: Superfolder Green Fluorescent Protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Aequorea victoria (jellyfish) |
Molecular weight | Theoretical: 26.623918 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT GKLPVPWPTL VTTL(CRO)VQCFS RYPDHM KRH DFFKSAMPEG YVQERTISFK DDGTYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNFNSHNVY ITADKQK NG IKANFKIRHN ...String: MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT GKLPVPWPTL VTTL(CRO)VQCFS RYPDHM KRH DFFKSAMPEG YVQERTISFK DDGTYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNFNSHNVY ITADKQK NG IKANFKIRHN VEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT HHHHHH |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 8 |
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Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 300 |
Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 33.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: AB INITIO MODEL |
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Output model | PDB-8g4e: |