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- EMDB-42845: Selenocysteine synthase- SelA -

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Basic information

Entry
Database: EMDB / ID: EMD-42845
TitleSelenocysteine synthase- SelA
Map dataSelA sharpened map (B-factor = 92.5 A^2)
Sample
  • Complex: Bacterial Selenocysteine Synthase
    • Protein or peptide: L-seryl-tRNA(Sec) selenium transferase
KeywordsSelenocysteine / tRNASec / Sec-synthase / RNA BINDING PROTEIN
Function / homology
Function and homology information


L-seryl-tRNASec selenium transferase / L-seryl-tRNA(Sec) selenium transferase activity / conversion of seryl-tRNAsec to selenocys-tRNAsec / selenocysteine biosynthetic process / selenocysteine incorporation / pyridoxal phosphate binding / identical protein binding / cytosol
Similarity search - Function
L-seryl-tRNA selenium transferase N-terminal domain / Selenocysteine synthase N terminal / L-seryl-tRNA(Sec) selenium transferase / L-seryl-tRNA selenium transferase-like / L-seryl-tRNA selenium transferase / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase
Similarity search - Domain/homology
L-seryl-tRNA(Sec) selenium transferase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.69 Å
AuthorsBalasco Serrao VH / Minari K / Pereira HM / Thiemann OH
Funding support Brazil, United States, 5 items
OrganizationGrant numberCountry
Sao Paulo Research Foundation (FAPESP)12/23730-1 Brazil
National Institutes of Health/National Center for Research Resources (NIH/NCRR)U24GM129547 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)S10OD02509 United States
Brazilian National Council for Scientific and Technological Development (CNPq)232251/2014-2 Brazil
Brazilian National Council for Scientific and Technological Development (CNPq)140636/2013-7 Brazil
CitationJournal: Curr Res Struct Biol / Year: 2024
Title: Bacterial selenocysteine synthase structure revealed by single-particle cryoEM.
Authors: Vitor Hugo Balasco Serrão / Karine Minari / Humberto D'Muniz Pereira / Otavio Henrique Thiemann /
Abstract: The 21st amino acid, selenocysteine (Sec), is synthesized on its dedicated transfer RNA (tRNA). In bacteria, Sec is synthesized from Ser-tRNA by Selenocysteine Synthase (SelA), which is a pivotal ...The 21st amino acid, selenocysteine (Sec), is synthesized on its dedicated transfer RNA (tRNA). In bacteria, Sec is synthesized from Ser-tRNA by Selenocysteine Synthase (SelA), which is a pivotal enzyme in the biosynthesis of Sec. The structural characterization of bacterial SelA is of paramount importance to decipher its catalytic mechanism and its role in the regulation of the Sec-synthesis pathway. Here, we present a comprehensive single-particle cryo-electron microscopy (SPA cryoEM) structure of the bacterial SelA with an overall resolution of 2.69 Å. Using recombinant SelA, we purified and prepared samples for single-particle cryoEM. The structural insights from SelA, combined with previous and knowledge, underscore the indispensable role of decamerization in SelA's function. Moreover, our structural analysis corroborates previous results that show that SelA adopts a pentamer of dimers configuration, and the active site architecture, substrate binding pocket, and key K295 catalytic residue are identified and described in detail. The differences in protein architecture and substrate coordination between the bacterial enzyme and its counterparts offer compelling structural evidence supporting the independent molecular evolution of the bacterial and archaea/eukarya Ser-Sec biosynthesis present in the natural world.
History
DepositionNov 16, 2023-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateJun 26, 2024-
Current statusJun 26, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42845.png

Downloads & links

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Map

FileDownload / File: emd_42845.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSelA sharpened map (B-factor = 92.5 A^2)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 480 pix.
= 396.48 Å		0.83 Å/pix.
x 480 pix.
= 396.48 Å		0.83 Å/pix.
x 480 pix.
= 396.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.826 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.75419116 - 1.1713678
Average (Standard dev.)0.00005636584 (±0.021423796)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 396.47998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map B

Fileemd_42845_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_42845_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Bacterial Selenocysteine Synthase

EntireName: Bacterial Selenocysteine Synthase
Components
  • Complex: Bacterial Selenocysteine Synthase
    • Protein or peptide: L-seryl-tRNA(Sec) selenium transferase

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Supramolecule #1: Bacterial Selenocysteine Synthase

SupramoleculeName: Bacterial Selenocysteine Synthase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Heterologously expressed E. coli homodecameric Selenocysteine Synthase
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12
Molecular weightTheoretical: 510 KDa

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Macromolecule #1: L-seryl-tRNA(Sec) selenium transferase

MacromoleculeName: L-seryl-tRNA(Sec) selenium transferase / type: protein_or_peptide / ID: 1 / Number of copies: 10 / Enantiomer: LEVO / EC number: L-seryl-tRNASec selenium transferase
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12
Molecular weightTheoretical: 50.895172 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTTETRSLYS QLPAIDRLLR DSSFLSLRDT YGHTRVVELL RQMLDEAREV IRGSQTLPAW CENWAQEVDA RLTKEAQSAL RPVINLTGT VLHTNLGRAL QAEAAVEAVA QAMRSPVTLE YDLDDAGRGH RDRALAQLLC RITGAEDACI VNNNAAAVLL M LAATASGK ...String:
MTTETRSLYS QLPAIDRLLR DSSFLSLRDT YGHTRVVELL RQMLDEAREV IRGSQTLPAW CENWAQEVDA RLTKEAQSAL RPVINLTGT VLHTNLGRAL QAEAAVEAVA QAMRSPVTLE YDLDDAGRGH RDRALAQLLC RITGAEDACI VNNNAAAVLL M LAATASGK EVVVSRGELV EIGGAFRIPD VMRQAGCTLH EVGTTNRTHA NDYRQAVNEN TALLMKVHTS NYSIQGFTKA ID EAELVAL GKELDVPVVT DLGSGSLVDL SQYGLPKEPM PQELIAAGVS LVSFSGD(LLP)LL GGPQAGIIVG KKEMIARLQ SHPLKRALRA DKMTLAALEA TLRLYLHPEA LSEKLPTLRL LTRSAEVIQI QAQRLQAPLA AHYGAEFAVQ VMPCLSQIGS GSLPVDRLP SAALTFTPHD GRGSHLESLA ARWRELPVPV IGRIYDGRLW LDLRCLEDEQ RFLEMLLK

UniProtKB: L-seryl-tRNA(Sec) selenium transferase

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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

Concentration3.9 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMKH2PO4Potassium Phosphate
100.0 mMNaClSodium Chloride
2.0 mM2-mercaptoethanol
10.0 uMpyridoxal 5-phosphate

Details: 20 mM potassium phosphate pH 7.5, 100 mM sodium chloride, 2 mM 2-mercaptoethanol, and 10 uM pyridoxal 5-phosphate.
GridModel: Quantifoil / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 20 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Pressure: 0.00035999999999999997 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
Details: The sample was blotted for 2.5 seconds using a force of -10 and then swiftly plunged frozen into liquid ethane using the Vitrobot Mark IV - Thermo Fisher Scientific..
DetailsMonodisperse sample after size-exclusion chromatography

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 4592 / Average electron dose: 45.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 688602
Startup modelType of model: NONE / Details: Ab initio reconstruction from selected particles.
Final reconstructionApplied symmetry - Point group: D5 (2x5 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.69 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 223410
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.2.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 223410 / Software - Name: cryoSPARC (ver. 4.2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 95.2
Output model

PDB-8uzw:
Selenocysteine synthase- SelA

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