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- PDB-8jgc: Cryo-EM structure of Mi3 fused with LOV2 -

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Basic information

Entry
Database: PDB / ID: 8jgc
TitleCryo-EM structure of Mi3 fused with LOV2
ComponentsLOV domain-containing protein,2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase
KeywordsLYASE / protein cage Scaffold
Function / homology
Function and homology information


D-galactonate catabolic process / 2-dehydro-3-deoxy-6-phosphogalactonate aldolase activity / response to blue light / photoreceptor activity / protein kinase activity / ATP binding / nucleus
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
LOV domain-containing protein / 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase
Similarity search - Component
Biological speciesAegilops tauschii subsp. strangulata (plant)
Thermotoga maritima (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.44 Å
AuthorsZhang, H.W. / Kang, W. / Xue, C.
Funding support China, 1items
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, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: LOV domain-containing protein,2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase


Theoretical massNumber of molelcules
Total (without water)41,4241
Polymers41,4241
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein LOV domain-containing protein,2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase


Mass: 41424.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aegilops tauschii subsp. strangulata (plant), (gene. exp.) Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) (bacteria)
Gene: TM_0066 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A453KFI0, UniProt: Q9WXS1
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: LOV2-Mi3 / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Thermotoga maritima (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: dev_3951: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 85541 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0081545
ELECTRON MICROSCOPYf_angle_d0.8942084
ELECTRON MICROSCOPYf_dihedral_angle_d6.18205
ELECTRON MICROSCOPYf_chiral_restr0.06244
ELECTRON MICROSCOPYf_plane_restr0.008263

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