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- EMDB-36230: Cryo-EM structure of Mi3 fused with LOV2 -

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Basic information

Entry
Database: EMDB / ID: EMD-36230
TitleCryo-EM structure of Mi3 fused with LOV2
Map data
Sample
  • Complex: LOV2-Mi3
    • Protein or peptide: LOV domain-containing protein,2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase
Keywordsprotein cage Scaffold / LYASE
Function / homology
Function and homology information


response to blue light / signaling / cell communication / photoreceptor activity / non-specific serine/threonine protein kinase / protein kinase activity / lyase activity / ATP binding
Similarity search - Function
KDPG/KHG aldolase / KDPG and KHG aldolase / PAS domain / PAS-associated, C-terminal / PAC domain profile. / PAC motif / Motif C-terminal to PAS motifs (likely to contribute to PAS structural domain) / PAS repeat profile. / PAS domain / PAS domain superfamily ...KDPG/KHG aldolase / KDPG and KHG aldolase / PAS domain / PAS-associated, C-terminal / PAC domain profile. / PAC motif / Motif C-terminal to PAS motifs (likely to contribute to PAS structural domain) / PAS repeat profile. / PAS domain / PAS domain superfamily / Aldolase-type TIM barrel / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
non-specific serine/threonine protein kinase / 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase
Similarity search - Component
Biological speciesThermotoga maritima (bacteria) / Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.44 Å
AuthorsZhang HW / Kang W / Xue C
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: J Am Chem Soc / Year: 2024
Title: Dynamic Metabolons Using Stimuli-Responsive Protein Cages.
Authors: Wei Kang / Xiao Ma / Huawei Zhang / Juncai Ma / Chunxue Liu / Jiani Li / Hanhan Guo / Daping Wang / Rui Wang / Bo Li / Chuang Xue /
Abstract: Naturally evolved metabolons have the ability to assemble and disassemble in response to environmental stimuli, allowing for the rapid reorganization of chemical reactions in living cells to meet ...Naturally evolved metabolons have the ability to assemble and disassemble in response to environmental stimuli, allowing for the rapid reorganization of chemical reactions in living cells to meet changing cellular needs. However, replicating such capability in synthetic metabolons remains a challenge due to our limited understanding of the mechanisms by which the assembly and disassembly of such naturally occurring multienzyme complexes are controlled. Here, we report the synthesis of chemical- and light-responsive protein cages for assembling synthetic metabolons, enabling the dynamic regulation of enzymatic reactions in living cells. Particularly, a chemically responsive domain was fused to a self-assembled protein cage subunit, generating engineered protein cages capable of displaying proteins containing cognate interaction domains on their surfaces in response to small molecular cues. Chemical-induced colocalization of sequential enzymes on protein cages enhances the specificity of the branched deoxyviolacein biosynthetic reactions by 2.6-fold. Further, by replacing the chemical-inducible domain with a light-inducible dimerization domain, we created an optogenetic protein cage capable of reversibly recruiting and releasing targeted proteins onto and from the exterior of the protein cages in tens of seconds by on-off of blue light. Tethering the optogenetic protein cages to membranes enables the formation of light-switchable, membrane-bound metabolons, which can repeatably recruit-release enzymes, leading to the manipulation of substrate utilization across membranes on demand. Our work demonstrates a powerful and versatile strategy for constructing dynamic metabolons in engineered living cells for efficient and controllable biocatalysis.
History
DepositionMay 20, 2023-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_36230.png

Downloads & links

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Map

FileDownload / File: emd_36230.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 512 pix.
= 437.76 Å		0.86 Å/pix.
x 512 pix.
= 437.76 Å		0.86 Å/pix.
x 512 pix.
= 437.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.855 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-0.50561583 - 0.78539133
Average (Standard dev.)0.002139045 (±0.027651155)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 437.76 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_36230_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_36230_half_map_2.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : LOV2-Mi3

EntireName: LOV2-Mi3
Components
  • Complex: LOV2-Mi3
    • Protein or peptide: LOV domain-containing protein,2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase

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Supramolecule #1: LOV2-Mi3

SupramoleculeName: LOV2-Mi3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thermotoga maritima (bacteria)

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Macromolecule #1: LOV domain-containing protein,2-dehydro-3-deoxyphosphogluconate a...

MacromoleculeName: LOV domain-containing protein,2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) (bacteria)
Molecular weightTheoretical: 41.424469 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSEFLATT LERIEKNFVI TDPRLPDNPI IFASDSFLQL TEYSREEILG RNCRFLQGPE TDRATVRKIR DAIDNQTEVT VQLINYTKS GKKFWNVFHL QPMRDYKGDV QYFIGVQLDG TERLHGAAER EAVCLIKKTA FQIAEAANDE NYFGGSGGSG G SGGSMKME ...String:
MGSSEFLATT LERIEKNFVI TDPRLPDNPI IFASDSFLQL TEYSREEILG RNCRFLQGPE TDRATVRKIR DAIDNQTEVT VQLINYTKS GKKFWNVFHL QPMRDYKGDV QYFIGVQLDG TERLHGAAER EAVCLIKKTA FQIAEAANDE NYFGGSGGSG G SGGSMKME ELFKKHKIVA VLRANSVEEA KKKALAVFLG GVHLIEITFT VPDADTVIKE LSFLKEMGAI IGAGTVTSVE QA RKAVESG AEFIVSPHLD EEISQFAKEK GVFYMPGVMT PTELVKAMKL GHTILKLFPG EVVGPQFVKA MKGPFPNVKF VPT GGVNLD NVCEWFKAGV LAVGVGSALV KGTPVEVAEK AKAFVEKIRG CTEGSGHHHH HH

UniProtKB: non-specific serine/threonine protein kinase, 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase

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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

BufferpH: 7.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7b3y

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 85541
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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