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- EMDB-28971: CryoEM map of de novo designed oligomeric protein C8-71_6x -

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Basic information

Entry
Database: EMDB / ID: EMD-28971
TitleCryoEM map of de novo designed oligomeric protein C8-71_6x
Map data
Sample
  • Complex: Self-assembled homo-octamer of de novo designed protein C8-71_6x
    • Protein or peptide: C8-71_6x
KeywordsSynthetic / Self-assembling / Oligomeric / Helical repeats / DE NOVO PROTEIN
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.0 Å
AuthorsRedler RL / Edman NI / Baker D / Ekiert DC / 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
CitationJournal: Cell / Year: 2024
Title: Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies.
Authors: Natasha I Edman / Ashish Phal / Rachel L Redler / Thomas Schlichthaerle / Sanjay R Srivatsan / Devon Duron Ehnes / Ali Etemadi / Seong J An / Andrew Favor / Zhe Li / Florian Praetorius / Max ...Authors: Natasha I Edman / Ashish Phal / Rachel L Redler / Thomas Schlichthaerle / Sanjay R Srivatsan / Devon Duron Ehnes / Ali Etemadi / Seong J An / Andrew Favor / Zhe Li / Florian Praetorius / Max Gordon / Thomas Vincent / Silvia Marchiano / Leslie Blakely / Chuwei Lin / 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 / Ying Zheng / Charles E Murry / Devin K Schweppe / Benjamin S Freedman / Lance Stewart / Damian C Ekiert / Joseph Schlessinger / Jay Shendure / Gira Bhabha / Hannele Ruohola-Baker / David Baker /
Abstract: Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase ...Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect 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 de novo-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 Ca release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.
History
DepositionNov 30, 2022-
Header (metadata) releaseJun 12, 2024-
Map releaseJun 12, 2024-
UpdateJun 26, 2024-
Current statusJun 26, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28971.png

Downloads & links

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Map

FileDownload / File: emd_28971.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.096 Å
Density
Contour LevelBy AUTHOR: 0.332
Minimum - Maximum-0.17662746 - 0.98054147
Average (Standard dev.)0.0018185998 (±0.047080167)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 280.576 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_28971_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_28971_half_map_2.map
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-octamer of de novo designed protein C8-71_6x

EntireName: Self-assembled homo-octamer of de novo designed protein C8-71_6x
Components
  • Complex: Self-assembled homo-octamer of de novo designed protein C8-71_6x
    • Protein or peptide: C8-71_6x

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Supramolecule #1: Self-assembled homo-octamer of de novo designed protein C8-71_6x

SupramoleculeName: Self-assembled homo-octamer of de novo designed protein C8-71_6x
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: C8-71_6x

MacromoleculeName: C8-71_6x / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MGPEEILERA KESLERAREA SERGDEEEFR KAAEKALELA KRLVEQAKKE GDPELVLEAA KVALRVAELA AKNGDKEVFK KAAESALEVA KRLVEVASKE GDPELVLEAA KVALEVARLA AENGDKEVFK KAAESALEVA KRLVEVASKE GDPELVLEAA RVALWVAELA ...String:
MGPEEILERA KESLERAREA SERGDEEEFR KAAEKALELA KRLVEQAKKE GDPELVLEAA KVALRVAELA AKNGDKEVFK KAAESALEVA KRLVEVASKE GDPELVLEAA KVALEVARLA AENGDKEVFK KAAESALEVA KRLVEVASKE GDPELVLEAA RVALWVAELA AKNGDKEVFK KAAESALEVA KRLVEVASKE GDPDLVAWAA LVALWVAFLA FLNGDKEVFK KAAESALEVA KALMEVAMKV GAPWLVELAI AVARAVWLLA ELFGDEEVRR RAEAFEIILR IAAIAVKAWL GGGGSLEHHH HHH

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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

Concentration0.1 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / 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: 263 / Average exposure time: 2.4 sec. / Average electron dose: 52.54 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 2.0 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: C8 (8 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 9092
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)

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