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- EMDB-47202: Structure of the native PLP synthase subunit PdxS from Methanosar... -

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Basic information

Entry
Database: EMDB / ID: EMD-47202
TitleStructure of the native PLP synthase subunit PdxS from Methanosarcina acetivorans
Map dataNative PLP synthase subunit PdxS from Methanosarcina acetivorans
Sample
  • Complex: Homo-dodecameric complex of the PdxS subunit of PLP synthase
    • Protein or peptide: Pyridoxal 5'-phosphate synthase subunit PdxS
Keywordspyridoxal 5'-phosphate synthase / TIM barrel / vitamer biosynthesis / BIOSYNTHETIC PROTEIN / LYASE
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsAgnew A / Humm E / Zhou K / Gunsalus R / Zhou ZH
Funding support United States, 5 items
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, 2024-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateJan 22, 2025-
Current statusJan 22, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47202.png

Downloads & links

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Map

FileDownload / File: emd_47202.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNative PLP synthase subunit PdxS from Methanosarcina acetivorans
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 300 pix.
= 330. Å		1.1 Å/pix.
x 300 pix.
= 330. Å		1.1 Å/pix.
x 300 pix.
= 330. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.403
Minimum - Maximum-2.5143228 - 3.144018
Average (Standard dev.)0.0003435644 (±0.08823045)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 330.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_47202_msk_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_47202_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_47202_half_map_2.map
AnnotationHalf Map A
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Homo-dodecameric complex of the PdxS subunit of PLP synthase

EntireName: Homo-dodecameric complex of the PdxS subunit of PLP synthase
Components
  • Complex: Homo-dodecameric complex of the PdxS subunit of PLP synthase
    • Protein or peptide: Pyridoxal 5'-phosphate synthase subunit PdxS

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Supramolecule #1: Homo-dodecameric complex of the PdxS subunit of PLP synthase

SupramoleculeName: Homo-dodecameric complex of the PdxS subunit of PLP synthase
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Native PdxS enriched from M. acetivorans lysate
Source (natural)Organism: Methanosarcina acetivorans C2A (archaea) / Strain: C2A
Molecular weightTheoretical: 387 KDa

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Macromolecule #1: Pyridoxal 5'-phosphate synthase subunit PdxS

MacromoleculeName: Pyridoxal 5'-phosphate synthase subunit PdxS / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
EC number: pyridoxal 5'-phosphate synthase (glutamine hydrolysing)
Source (natural)Organism: Methanosarcina acetivorans C2A (archaea) / Strain: C2A
Molecular weightTheoretical: 30.986889 KDa
SequenceString: HGTELIKRGF ARMQKGGVIM DVTTPEQARI AEEAGAVAVM ALQAVPADIR KAGGVARMAD PEIVQQIIET VTIPVMAKAR IGHFVEAEI LEALGVDMVD ESEVLTPADP FYHIDKTQFT VPFVCGARNL GEALRRINEG AAMIRTKGEA GTGDVSQAVK H MKQIQGEI ...String:
HGTELIKRGF ARMQKGGVIM DVTTPEQARI AEEAGAVAVM ALQAVPADIR KAGGVARMAD PEIVQQIIET VTIPVMAKAR IGHFVEAEI LEALGVDMVD ESEVLTPADP FYHIDKTQFT VPFVCGARNL GEALRRINEG AAMIRTKGEA GTGDVSQAVK H MKQIQGEI RALAGKTKEE LIMVAREIEA PIELVVETAK MQRLPVVNFA AGGVATPADA ALMMRLGADG VFVGSGIFKA EN PEKMAKA VVEAVNNYDN PVKLAEISKG VGAGMKGISA DMIPAQEALQ ERGW

UniProtKB: Pyridoxal 5'-phosphate synthase subunit PdxS

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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: 7.4
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was heterogeneous; multiple other proteins were present on grids.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 99971
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6-f1


Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementOverall B value: 157
Output model

PDB-9dvf:
Structure of the native PLP synthase subunit PdxS from Methanosarcina acetivorans

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