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- PDB-9dvf: Structure of the native PLP synthase subunit PdxS from Methanosar... -

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Basic information

Entry
Database: PDB / ID: 9dvf
TitleStructure of the native PLP synthase subunit PdxS from Methanosarcina acetivorans
ComponentsPyridoxal 5'-phosphate synthase subunit PdxS
KeywordsLYASE / pyridoxal 5'-phosphate synthase / TIM barrel / vitamer biosynthesis / BIOSYNTHETIC PROTEIN
Function / homology
Function and homology information


amine-lyase activity / pyridoxal 5'-phosphate synthase (glutamine hydrolysing) / pyridoxal 5'-phosphate synthase (glutamine hydrolysing) activity / pyridoxal phosphate biosynthetic process / pyridoxine biosynthetic process / amino acid metabolic process
Similarity search - Function
Pyridoxal 5'-phosphate synthase subunit PdxS/SNZ / PdxS/SNZ N-terminal domain / SOR/SNZ family / PdxS/SNZ family signature. / PdxS/SNZ family profile. / Ribulose-phosphate binding barrel / Aldolase-type TIM barrel
Similarity search - Domain/homology
Pyridoxal 5'-phosphate synthase subunit PdxS
Similarity search - Component
Biological speciesMethanosarcina acetivorans C2A (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsAgnew, A. / Humm, E. / Zhou, K. / Gunsalus, R. / Zhou, Z.H.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM071940 United States
National Science Foundation (NSF, United States)1911781 United States
Department of Energy (DOE, United States)DE-FC-02-02ER63421 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM145388 United States
National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR)T90DE030860 United States
CitationJournal: mBio / Year: 2025
Title: Structure and identification of the native PLP synthase complex from lysate.
Authors: Angela Agnew / Ethan Humm / Kang Zhou / Robert P Gunsalus / Z Hong Zhou /
Abstract: Many protein-protein interactions behave differently in biochemically purified forms as compared to their states. As such, determining native protein structures may elucidate structural states ...Many protein-protein interactions behave differently in biochemically purified forms as compared to their states. As such, determining native protein structures may elucidate structural states previously unknown for even well-characterized proteins. Here, we apply the bottom-up structural proteomics method, , toward a model methanogenic archaeon. While they are keystone organisms in the global carbon cycle and active members of the human microbiome, there is a general lack of characterization of methanogen enzyme structure and function. Through the approach, we successfully reconstructed and identified the native pyridoxal 5'-phosphate (PLP) synthase (PdxS) complex directly from cryogenic electron microscopy (cryo-EM) images of fractionated cellular lysate. We found that the native PdxS complex exists as a homo-dodecamer of PdxS subunits, and the previously proposed supracomplex containing both the synthase (PdxS) and glutaminase (PdxT) was not observed in cellular lysate. Our structure shows that the native PdxS monomer fashions a single 8α/8β TIM-barrel domain, surrounded by seven additional helices to mediate solvent and interface contacts. A density is present at the active site in the cryo-EM map and is interpreted as ribose 5-phosphate. In addition to being the first reconstruction of the PdxS enzyme from a heterogeneous cellular sample, our results reveal a departure from previously published archaeal PdxS crystal structures, lacking the 37-amino-acid insertion present in these prior cases. This study demonstrates the potential of applying the workflow to capture native structural states at atomic resolution for archaeal systems, for which traditional biochemical sample preparation is nontrivial.IMPORTANCEArchaea are one of the three domains of life, classified as a phylogenetically distinct lineage. There is a paucity of known enzyme structures from organisms of this domain, and this is often exacerbated by characteristically difficult growth conditions and a lack of readily available molecular biology toolkits to study proteins in archaeal cells. As a result, there is a gap in knowledge concerning the mechanisms governing archaeal protein behavior and their impacts on both the environment and human health; case in point, the synthesis of the widely utilized cofactor pyridoxal 5'-phosphate (PLP; a vitamer of vitamin B6, which humans cannot produce). By leveraging the power of single-particle cryo-EM and map-to-primary sequence identification, we determine the native structure of PLP synthase from cellular lysate. Our workflow allows the (i) rapid examination of new or less characterized systems with minimal sample requirements and (ii) discovery of structural states inaccessible by recombinant expression.
History
DepositionOct 7, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Jan 22, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pyridoxal 5'-phosphate synthase subunit PdxS
B: Pyridoxal 5'-phosphate synthase subunit PdxS
C: Pyridoxal 5'-phosphate synthase subunit PdxS
D: Pyridoxal 5'-phosphate synthase subunit PdxS
E: Pyridoxal 5'-phosphate synthase subunit PdxS
F: Pyridoxal 5'-phosphate synthase subunit PdxS
G: Pyridoxal 5'-phosphate synthase subunit PdxS
H: Pyridoxal 5'-phosphate synthase subunit PdxS
I: Pyridoxal 5'-phosphate synthase subunit PdxS
J: Pyridoxal 5'-phosphate synthase subunit PdxS
K: Pyridoxal 5'-phosphate synthase subunit PdxS
L: Pyridoxal 5'-phosphate synthase subunit PdxS


Theoretical massNumber of molelcules
Total (without water)371,84312
Polymers371,84312
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Pyridoxal 5'-phosphate synthase subunit PdxS / PLP synthase subunit PdxS / Pdx1


Mass: 30986.889 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Source: (natural) Methanosarcina acetivorans C2A (archaea) / Strain: C2A
References: UniProt: Q8TQH6, pyridoxal 5'-phosphate synthase (glutamine hydrolysing)
Has protein modificationN

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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: Homo-dodecameric complex of the PdxS subunit of PLP synthase
Type: COMPLEX / Details: Native PdxS enriched from M. acetivorans lysate / Entity ID: all / Source: NATURAL
Molecular weightValue: 0.387 MDa / Experimental value: NO
Source (natural)Organism: Methanosarcina acetivorans C2A (archaea) / Strain: C2A
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: This sample was heterogeneous; multiple other proteins were present on grids.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1800 nm
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2SerialEM4.0.26image acquisition
4cryoSPARC4.3.1CTF correction
7ISOLDEmodel fitting
10cryoSPARC4.3.1final Euler assignment
11cryoSPARC4.3.1classification
12cryoSPARC4.3.13D reconstruction
19PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D6 (2x6 fold dihedral)
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 99971 / Symmetry type: POINT
Atomic model buildingB value: 157
Atomic model buildingAccession code: AF-Q8TQH6-F1 / Source name: AlphaFold / Type: in silico model

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