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- EMDB-28958: CryoEM structure of designed modular protein oligomer C4-131 -

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Basic information

Entry
Database: EMDB / ID: EMD-28958
TitleCryoEM structure of designed modular protein oligomer C4-131
Map datadesigned modular protein oligomer C4-131
Sample
  • Complex: Self-assembled homo-tetramer of de novo designed protein C4-131
    • Protein or peptide: C4-131
KeywordsSynthetic / Self-assembling / Oligomeric / Helical repeats / DE NOVO PROTEIN
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.1 Å
AuthorsRedler RL / Edman NI / Baker D / Ekiert D / Bhabha G
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM128777 United States
National Institutes of Health/National Institute on Aging (NIH/NIA)R01AG063845 United States
Citation
Journal: bioRxiv / Year: 2023
Title: Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies.
Authors: Natasha I Edman / Rachel L Redler / Ashish Phal / Thomas Schlichthaerle / Sanjay R Srivatsan / Ali Etemadi / Seong J An / Andrew Favor / Devon Ehnes / Zhe Li / Florian Praetorius / Max ...Authors: Natasha I Edman / Rachel L Redler / Ashish Phal / Thomas Schlichthaerle / Sanjay R Srivatsan / Ali Etemadi / Seong J An / Andrew Favor / Devon Ehnes / Zhe Li / Florian Praetorius / Max Gordon / Wei Yang / Brian Coventry / Derrick R Hicks / Longxing Cao / Neville Bethel / Piper Heine / Analisa Murray / Stacey Gerben / Lauren Carter / Marcos Miranda / Babak Negahdari / Sangwon Lee / Cole Trapnell / Lance Stewart / Damian C Ekiert / Joseph Schlessinger / Jay Shendure / Gira Bhabha / Hannele Ruohola-Baker / David Baker /
Abstract: Growth factors and cytokines signal by binding to the extracellular domains of their receptors and drive association and transphosphorylation of the receptor intracellular tyrosine kinase domains, ...Growth factors and cytokines signal by binding to the extracellular domains of their receptors and drive association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affects signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a designed fibroblast growth-factor receptor (FGFR) binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and MAPK pathway activation. The high specificity of the designed agonists reveal distinct roles for two FGFR splice variants in driving endothelial and mesenchymal cell fates during early vascular development. The ability to incorporate receptor binding domains and repeat extensions in a modular fashion makes our designed scaffolds broadly useful for probing and manipulating cellular signaling pathways.
#2: Journal: Cell(Cambridge,Mass.) / Year: 2024
Title: Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies
Authors: Edman NI / Redler RL / Phal A / Schlichthaerle T / Srivatsan SR / Etemadi A / An SJ / Favor A / Ehnes D / Li Z / Praetorius F / Gordon M / Yang W / Coventry B / Hicks DR / Cao L / Bethel N / ...Authors: Edman NI / Redler RL / Phal A / Schlichthaerle T / Srivatsan SR / Etemadi A / An SJ / Favor A / Ehnes D / Li Z / Praetorius F / Gordon M / Yang W / Coventry B / Hicks DR / Cao L / Bethel N / Heine P / Murray A / Gerben S / Carter L / Miranda M / Negahdari B / Lee S / Trapnell C / Stewart L / Ekiert DC / Schlessinger J / Shendure J / Bhabha G / Ruohola-Baker H / Baker D
History
DepositionNov 28, 2022-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateJun 26, 2024-
Current statusJun 26, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28958.png

Downloads & links

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Map

FileDownload / File: emd_28958.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationdesigned modular protein oligomer C4-131
Voxel sizeX=Y=Z: 1.096 Å
Density
Contour LevelBy AUTHOR: 0.248
Minimum - Maximum-0.28990415 - 1.1213306
Average (Standard dev.)0.005208059 (±0.056276545)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 164.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_28958_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_28958_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 : Self-assembled homo-tetramer of de novo designed protein C4-131

EntireName: Self-assembled homo-tetramer of de novo designed protein C4-131
Components
  • Complex: Self-assembled homo-tetramer of de novo designed protein C4-131
    • Protein or peptide: C4-131

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Supramolecule #1: Self-assembled homo-tetramer of de novo designed protein C4-131

SupramoleculeName: Self-assembled homo-tetramer of de novo designed protein C4-131
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: C4-131

MacromoleculeName: C4-131 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MGLKELLKRA EELAKSPDPE DLKEAVRLAE EVVRERPGSE AAKKALEIIQ EAAEKLKKSP DPEAIIAAAR ALLKIAATTG DNEAAKQAIE AASKAAQLAE QRGDDELVCE ALALLIAAQV LLLKQQGVPM LEVAIHVAET ILQILQRLKR KGASEEVRKE CLKRILREIA ...String:
MGLKELLKRA EELAKSPDPE DLKEAVRLAE EVVRERPGSE AAKKALEIIQ EAAEKLKKSP DPEAIIAAAR ALLKIAATTG DNEAAKQAIE AASKAAQLAE QRGDDELVCE ALALLIAAQV LLLKQQGVPM LEVAIHVAET ILQILQRLKR KGASEEVRKE CLKRILREIA EALQRSGVPE EEIALIMLLI ILLLMMLGSL EHHHHHH

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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

Concentration0.75 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: HOLEY ARRAY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: CONTINUOUS / Support film - #1 - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 5 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: blot time = 4s; blot force = 0.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 26 / Average exposure time: 2.8 sec. / Average electron dose: 56.91 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.9 µm / Nominal defocus min: 2.1 µm / Nominal magnification: 36000
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: Ab initio model generated in Cryosparc
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 13986
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)

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