[English] 日本語
Yorodumi
- EMDB-29718: Green Fluorescence Protein imaged on a cryo-EM imaging scaffold -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-29718
TitleGreen Fluorescence Protein imaged on a cryo-EM imaging scaffold
Map dataGreen Fluorescence Protein imaged on a cryo-EM imaging scaffold
Sample
  • Complex: sfGFP displayed on a Cryo-EM imaging scaffold
    • Protein or peptide: RCG-10 - Cryo-EM imaging scaffold subunit B fused to DARPin
    • Protein or peptide: Superfolder Green Fluorescent Protein
KeywordsCryoEM imaging scaffold / Cancer / GTPase / SIGNALING PROTEIN
Biological speciessynthetic construct (others) / Aequorea victoria (jellyfish)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.98 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-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: RCSB / Status: Released

-
Structure visualization

Supplemental images

emd_29718.png

Downloads & links

-
Map

FileDownload / File: emd_29718.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGreen Fluorescence Protein imaged on a cryo-EM imaging scaffold
Voxel sizeX=Y=Z: 1.2375 Å
Density
Contour LevelBy AUTHOR: 0.7
Minimum - Maximum-4.8770094 - 7.0146117
Average (Standard dev.)0.013487803 (±0.08067675)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 316.8 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Half map: Half Map 1

Fileemd_29718_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: Half Map 2

Fileemd_29718_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : sfGFP displayed on a Cryo-EM imaging scaffold

EntireName: sfGFP displayed on a Cryo-EM imaging scaffold
Components
  • Complex: sfGFP displayed on a Cryo-EM imaging scaffold
    • Protein or peptide: RCG-10 - Cryo-EM imaging scaffold subunit B fused to DARPin
    • Protein or peptide: Superfolder Green Fluorescent Protein

-
Supramolecule #1: sfGFP displayed on a Cryo-EM imaging scaffold

SupramoleculeName: sfGFP displayed on a Cryo-EM imaging scaffold / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)

-
Macromolecule #1: RCG-10 - Cryo-EM imaging scaffold subunit B fused to DARPin

MacromoleculeName: RCG-10 - 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: 35.287246 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 YDDQVAALLA KGADVNAADD VGVTPLHLAA QRGHLEIVEV LLKRGADINA ADLWGQTPLH LA ATAGHLE IVELLLRWGA DVNARDNIGH TPLHLAAWAG HLEIVEVLLK YGADVNAQDK FGKTPFDLAI DNGNEDIAEV LQK AA

-
Macromolecule #2: Superfolder Green Fluorescent Protein

MacromoleculeName: Superfolder Green Fluorescent Protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Aequorea victoria (jellyfish)
Molecular weightTheoretical: 26.623918 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

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

-
Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 33.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

-
Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1221977
FSC plot (resolution estimation)

-
Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8g4e:
Green Fluorescence Protein imaged on a cryo-EM imaging scaffold

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more