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- EMDB-36808: Cryo-EM structure of KEOPS complex from Arabidopsis thaliana -

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Basic information

Entry
Database: EMDB / ID: EMD-36808
TitleCryo-EM structure of KEOPS complex from Arabidopsis thaliana
Map dataElectron density map for KEOPS complex from Arabidopsis thaliana
Sample
  • Complex: KEOPS complex from Arabidopsis thaliana
    • Protein or peptide: Probable tRNA N6-adenosine threonylcarbamoyltransferase
    • Protein or peptide: non-specific serine/threonine protein kinaseSerine/threonine-specific protein kinase
    • Protein or peptide: At4g34412
    • Protein or peptide: At5g53043
  • Ligand: FE (III) ION
KeywordsKEOPS complex / tRNA t6A synthase / RNA-binding protein / TRANSFERASE
Function / homology
Function and homology information


N(6)-L-threonylcarbamoyladenine synthase activity / EKC/KEOPS complex / tRNA threonylcarbamoyladenosine modification / tRNA processing / non-specific serine/threonine protein kinase / phosphorylation / protein serine/threonine kinase activity / ATP binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
tRNA N6-adenosine threonylcarbamoyltransferase Kae1, archaea and eukaryote / Serine/threonine-protein kinase Bud32 / CGI121/TPRKB / CGI121/TPRKB superfamily / Kinase binding protein CGI-121 / CTAG/Pcc1 family / Transcription factor Pcc1 / Kae1/TsaD family / Gcp-like domain / tRNA N6-adenosine threonylcarbamoyltransferase ...tRNA N6-adenosine threonylcarbamoyltransferase Kae1, archaea and eukaryote / Serine/threonine-protein kinase Bud32 / CGI121/TPRKB / CGI121/TPRKB superfamily / Kinase binding protein CGI-121 / CTAG/Pcc1 family / Transcription factor Pcc1 / Kae1/TsaD family / Gcp-like domain / tRNA N6-adenosine threonylcarbamoyltransferase / ATPase, nucleotide binding domain / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Glycoprotease 2 / At4g34412 / At5g53043 / non-specific serine/threonine protein kinase
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsZheng XX / Zhu L / Duan L / Zhang WH
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32000847 China
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Molecular basis of A. thaliana KEOPS complex in biosynthesizing tRNA t6A.
Authors: Xinxing Zheng / Chenchen Su / Lei Duan / Mengqi Jin / Yongtao Sun / Li Zhu / Wenhua Zhang /
Abstract: In archaea and eukaryotes, the evolutionarily conserved KEOPS is composed of four core subunits-Kae1, Bud32, Cgi121 and Pcc1, and a fifth Gon7/Pcc2 that is found in fungi and metazoa. KEOPS ...In archaea and eukaryotes, the evolutionarily conserved KEOPS is composed of four core subunits-Kae1, Bud32, Cgi121 and Pcc1, and a fifth Gon7/Pcc2 that is found in fungi and metazoa. KEOPS cooperates with Sua5/YRDC to catalyze the biosynthesis of tRNA N6-threonylcarbamoyladenosine (t6A), an essential modification needed for fitness of cellular organisms. Biochemical and structural characterizations of KEOPSs from archaea, yeast and humans have determined a t6A-catalytic role for Kae1 and auxiliary roles for other subunits. However, the precise molecular workings of KEOPSs still remain poorly understood. Here, we investigated the biochemical functions of A. thaliana KEOPS and determined a cryo-EM structure of A. thaliana KEOPS dimer. We show that A. thaliana KEOPS is composed of KAE1, BUD32, CGI121 and PCC1, which adopts a conserved overall arrangement. PCC1 dimerization leads to a KEOPS dimer that is needed for an active t6A-catalytic KEOPS-tRNA assembly. BUD32 participates in direct binding of tRNA to KEOPS and modulates the t6A-catalytic activity of KEOPS via its C-terminal tail and ATP to ADP hydrolysis. CGI121 promotes the binding of tRNA to KEOPS and potentiates the t6A-catalytic activity of KEOPS. These data and findings provide insights into mechanistic understanding of KEOPS machineries.
History
DepositionJul 11, 2023-
Header (metadata) releaseApr 3, 2024-
Map releaseApr 3, 2024-
UpdateApr 10, 2024-
Current statusApr 10, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36808.png

Downloads & links

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Map

FileDownload / File: emd_36808.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationElectron density map for KEOPS complex from Arabidopsis thaliana
Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.27099746 - 0.52531534
Average (Standard dev.)-0.00016466905 (±0.0065891924)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 440.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A for KEOPS complex from Arabidopsis thaliana

Fileemd_36808_half_map_1.map
AnnotationHalf map A for KEOPS complex from Arabidopsis thaliana
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B for KEOPS complex from Arabidopsis thaliana

Fileemd_36808_half_map_2.map
AnnotationHalf map B for KEOPS complex from Arabidopsis thaliana
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : KEOPS complex from Arabidopsis thaliana

EntireName: KEOPS complex from Arabidopsis thaliana
Components
  • Complex: KEOPS complex from Arabidopsis thaliana
    • Protein or peptide: Probable tRNA N6-adenosine threonylcarbamoyltransferase
    • Protein or peptide: non-specific serine/threonine protein kinaseSerine/threonine-specific protein kinase
    • Protein or peptide: At4g34412
    • Protein or peptide: At5g53043
  • Ligand: FE (III) ION

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Supramolecule #1: KEOPS complex from Arabidopsis thaliana

SupramoleculeName: KEOPS complex from Arabidopsis thaliana / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: The sample contains KAE1, BUD32, CGI121 and PCC1, which form a KEOPS complex.
Source (natural)Organism: Arabidopsis thaliana (thale cress)

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Macromolecule #1: Probable tRNA N6-adenosine threonylcarbamoyltransferase

MacromoleculeName: Probable tRNA N6-adenosine threonylcarbamoyltransferase
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: N6-L-threonylcarbamoyladenine synthase
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 38.81357 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKKMIAIGF EGSANKIGVG IVTLDGTILA NPRHTYITPP GHGFLPRETA HHHLDHVLPL VKSALETSQV TPEEIDCICY TKGPGMGAP LQVSAIVVRV LSQLWKKPIV AVNHCVAHIE MGRVVTGADD PVVLYVSGGN TQVIAYSEGR YRIFGETIDI A VGNCLDRF ...String:
MKKKMIAIGF EGSANKIGVG IVTLDGTILA NPRHTYITPP GHGFLPRETA HHHLDHVLPL VKSALETSQV TPEEIDCICY TKGPGMGAP LQVSAIVVRV LSQLWKKPIV AVNHCVAHIE MGRVVTGADD PVVLYVSGGN TQVIAYSEGR YRIFGETIDI A VGNCLDRF ARVLKLSNDP SPGYNIEQLA KKGENFIDLP YAVKGMDVSF SGILSYIETT AEEKLKNNEC TPADLCYSLQ ET VFAMLVE ITERAMAHCD KKDVLIVGGV GCNERLQEMM RTMCSERDGK LFATDDRYCI DNGAMIAYTG LLAFVNGIET PIE DSTFTQ RFRTDEVHAV WREKEAVLLG DKKVAAN

UniProtKB: Glycoprotease 2

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Macromolecule #2: non-specific serine/threonine protein kinase

MacromoleculeName: non-specific serine/threonine protein kinase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 25.170117 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDCEENVRDE SLVLIKQGAE ARVFESTFAG RRSIVKERFS KKYRHPILDA KLTLKRLNAE ARCMTKARKL GVCTPVLYAV DTLLHSLTL EYIEGVSVKD IFLEFGTNGV VEERLDDVAA QIGAAIAKLH DGGLAHGDLT TSNMLVRSGT NQLVLIDFGL S VTSTLPED ...String:
MDCEENVRDE SLVLIKQGAE ARVFESTFAG RRSIVKERFS KKYRHPILDA KLTLKRLNAE ARCMTKARKL GVCTPVLYAV DTLLHSLTL EYIEGVSVKD IFLEFGTNGV VEERLDDVAA QIGAAIAKLH DGGLAHGDLT TSNMLVRSGT NQLVLIDFGL S VTSTLPED KAVDLYVLER ALLSMHSSCG NVMDRILTAY RKSSKQWSAT FNKLAQVRQR GRKRTMIG

UniProtKB: non-specific serine/threonine protein kinase

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Macromolecule #3: At4g34412

MacromoleculeName: At4g34412 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 19.678484 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MKVFNLDRGN TLSVSLFSGV TNSKELLNSM LDGSLKLEVS FLNASLIPDI FPLLAAAQKA LISKSRDSLS TRTLHSELVY NYSGSKHIT ESLKRCGISE NTTYILAARF NASPVEMEEV AKLINGKEID LEELKTHANQ ANILKHYKIT SQELGISSLG D AIVCRIAA RDALHHHHHH

UniProtKB: At4g34412

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Macromolecule #4: At5g53043

MacromoleculeName: At5g53043 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 11.592994 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAATVPATTR SDQTWDFSCN LDVSFESEEH ALIAYTSLAV DKELQPDKVR RVMSVSNNKL SVHFEAIEAR LLRASFSAFV DVLTLATRT IQEFGQKHHH HHH

UniProtKB: At5g53043

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Macromolecule #5: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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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: 7.5
Details: 300 mM NaCl, 20 mM Tris-HCl pH 7.5,5 mM 2-mercaptoethanol
VitrificationCryogen name: ETHANE
DetailsThe sample was freshly purified by size exclusion chromatography.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.27 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
TemperatureMin: 77.1 K / Max: 89.8 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Average exposure time: 3.6 sec. / Average electron dose: 64.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 232232
FSC plot (resolution estimation)

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