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- EMDB-46630: Structure of the KEOPS complex (Cgi121/Bud32/Kae1/Pcc1) bound to ... -

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Entry
Database: EMDB / ID: EMD-46630
TitleStructure of the KEOPS complex (Cgi121/Bud32/Kae1/Pcc1) bound to tRNA in a distorted tRNA conformation
Map dataStructure of the KEOPS complex (Cgi121/Bud32/Kae1/Pcc1) bound to tRNA in a distorted tRNA conformation
Sample
  • Complex: Structure of the KEOPS complex (Cgi121/Bud32/Kae1/Pcc1)
    • Complex: Bud32
      • Protein or peptide: Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein
    • Complex: Cgi121
      • Protein or peptide: Regulatory protein Cgi121
    • Complex: Kae1
      • Protein or peptide: Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein
    • Complex: Pcc1
    • Protein or peptide: KEOPS complex subunit Pcc1
    • RNA: tRNA
KeywordstRNA / modification / t6A / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


N6-L-threonylcarbamoyladenine synthase / N(6)-L-threonylcarbamoyladenine synthase activity / EKC/KEOPS complex / tRNA threonylcarbamoyladenosine modification / protein serine/threonine/tyrosine kinase activity / metalloendopeptidase activity / 3-phosphoinositide-dependent protein kinase activity / DNA-dependent protein kinase activity / ribosomal protein S6 kinase activity / histone H3S10 kinase activity ...N6-L-threonylcarbamoyladenine synthase / N(6)-L-threonylcarbamoyladenine synthase activity / EKC/KEOPS complex / tRNA threonylcarbamoyladenosine modification / protein serine/threonine/tyrosine kinase activity / metalloendopeptidase activity / 3-phosphoinositide-dependent protein kinase activity / DNA-dependent protein kinase activity / ribosomal protein S6 kinase activity / histone H3S10 kinase activity / histone H2AXS139 kinase activity / histone H3S28 kinase activity / histone H4S1 kinase activity / histone H2BS14 kinase activity / histone H3T3 kinase activity / histone H2AS121 kinase activity / Rho-dependent protein serine/threonine kinase activity / histone H2BS36 kinase activity / histone H3S57 kinase activity / histone H2AT120 kinase activity / AMP-activated protein kinase activity / histone H2AS1 kinase activity / histone H3T6 kinase activity / histone H3T11 kinase activity / histone H3T45 kinase activity / non-specific serine/threonine protein kinase / iron ion binding / protein serine kinase activity / zinc ion binding / ATP binding / cytoplasm
Similarity search - Function
: / Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein / tRNA N6-adenosine threonylcarbamoyltransferase Kae1, archaea and eukaryote / Serine/threonine-protein kinase Bud32 / CGI121/TPRKB / CGI121/TPRKB superfamily / Kinase binding protein CGI-121 / Peptidase M22, conserved site / Glycoprotease family signature. / Kae1/TsaD family ...: / Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein / tRNA N6-adenosine threonylcarbamoyltransferase Kae1, archaea and eukaryote / Serine/threonine-protein kinase Bud32 / CGI121/TPRKB / CGI121/TPRKB superfamily / Kinase binding protein CGI-121 / Peptidase M22, conserved site / Glycoprotease family signature. / Kae1/TsaD family / CTAG/Pcc1 family / Transcription factor Pcc1 / Gcp-like domain / tRNA N6-adenosine threonylcarbamoyltransferase / RIO domain / ATPase, nucleotide binding domain / Tyrosine protein kinases specific active-site signature. / Tyrosine-protein kinase, active site / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Regulatory protein Cgi121 / Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein / KEOPS complex subunit Pcc1
Similarity search - Component
Biological speciesMethanocaldococcus jannaschii (archaea) / Pyrococcus furiosus (archaea) / Pyrococcus furiosus DSM 3638 (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.59 Å
AuthorsOna Chuquimarca SM / Beenstock J / Daou S / Keszei AFA / Yin Z / Sicheri F
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT-178026 Canada
CitationJournal: Nat Commun / Year: 2024
Title: Structures of KEOPS bound to tRNA reveal functional roles of the kinase Bud32.
Authors: Samara Mishelle Ona Chuquimarca / Jonah Beenstock / Salima Daou / Jennifer Porat / Alexander F A Keszei / Jay Z Yin / Tobias Beschauner / Mark A Bayfield / Mohammad T Mazhab-Jafari / Frank Sicheri /
Abstract: The enzyme complex KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) installs the universally conserved and essential N-threonylcarbamoyl adenosine modification (tA) on ANN-decoding ...The enzyme complex KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) installs the universally conserved and essential N-threonylcarbamoyl adenosine modification (tA) on ANN-decoding tRNAs in eukaryotes and in archaea. KEOPS consists of Cgi121, Kae1, Pcc1, Gon7 and the atypical kinase/ATPase Bud32. Except Gon7, all KEOPS subunits are needed for tRNA modification, and in humans, mutations in all five genes underlie the lethal genetic disease Galloway Mowat Syndrome (GAMOS). Kae1 catalyzes the modification of tRNA, but the specific contributions of Bud32 and the other subunits are less clear. Here we solved cryogenic electron microscopy structures of KEOPS with and without a tRNA substrate. We uncover distinct flexibility of KEOPS-bound tRNA revealing a conformational change that may enable its modification by Kae1. We further identified a contact between a flipped-out base of the tRNA and an arginine residue in C-terminal tail of Bud32 that correlates with the conformational change in the tRNA. We also uncover contact surfaces within the KEOPS-tRNA holo-enzyme substrate complex that are required for Bud32 ATPase regulation and tA modification activity. Our findings uncover inner workings of KEOPS including a basis for substrate specificity and why Kae1 depends on all other subunits.
History
DepositionAug 18, 2024-
Header (metadata) releaseDec 18, 2024-
Map releaseDec 18, 2024-
UpdateDec 25, 2024-
Current statusDec 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46630.png

Downloads & links

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Map

FileDownload / File: emd_46630.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the KEOPS complex (Cgi121/Bud32/Kae1/Pcc1) bound to tRNA in a distorted tRNA conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 256 pix.
= 263.68 Å		1.03 Å/pix.
x 256 pix.
= 263.68 Å		1.03 Å/pix.
x 256 pix.
= 263.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.321
Minimum - Maximum-1.5199294 - 2.2214818
Average (Standard dev.)0.000039594594 (±0.04451182)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 263.68 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map A

Fileemd_46630_half_map_1.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_46630_half_map_2.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of the KEOPS complex (Cgi121/Bud32/Kae1/Pcc1)

EntireName: Structure of the KEOPS complex (Cgi121/Bud32/Kae1/Pcc1)
Components
  • Complex: Structure of the KEOPS complex (Cgi121/Bud32/Kae1/Pcc1)
    • Complex: Bud32
      • Protein or peptide: Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein
    • Complex: Cgi121
      • Protein or peptide: Regulatory protein Cgi121
    • Complex: Kae1
      • Protein or peptide: Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein
    • Complex: Pcc1
    • Protein or peptide: KEOPS complex subunit Pcc1
    • RNA: tRNA

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Supramolecule #1: Structure of the KEOPS complex (Cgi121/Bud32/Kae1/Pcc1)

SupramoleculeName: Structure of the KEOPS complex (Cgi121/Bud32/Kae1/Pcc1)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Methanocaldococcus jannaschii (archaea)

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Supramolecule #2: Bud32

SupramoleculeName: Bud32 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Methanocaldococcus jannaschii (archaea)

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Supramolecule #3: Cgi121

SupramoleculeName: Cgi121 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Methanocaldococcus jannaschii (archaea)

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Supramolecule #4: Kae1

SupramoleculeName: Kae1 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Methanocaldococcus jannaschii (archaea)

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Supramolecule #5: Pcc1

SupramoleculeName: Pcc1 / type: complex / ID: 5 / Parent: 1
Source (natural)Organism: Pyrococcus furiosus (archaea)

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Macromolecule #1: Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesi...

MacromoleculeName: Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Methanocaldococcus jannaschii (archaea)
Molecular weightTheoretical: 23.957547 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: KIPEHLIGKG AEADIKRDSY LDFDVIIKER VKKGYRDERL DENIRKSRTA REARYLALVK DFGIPAPYIF DVDLDNKRIM MSYINGKLA KDVIEDNLDI AYKIGEIVGK LHKNDVIHND LTTSNFIFDK DLYIIDFGLG KISNLDRDKA VDLIVFKKAV L STHHEKFD ...String:
KIPEHLIGKG AEADIKRDSY LDFDVIIKER VKKGYRDERL DENIRKSRTA REARYLALVK DFGIPAPYIF DVDLDNKRIM MSYINGKLA KDVIEDNLDI AYKIGEIVGK LHKNDVIHND LTTSNFIFDK DLYIIDFGLG KISNLDRDKA VDLIVFKKAV L STHHEKFD EIWERFLEGY KSVYDRWEII LELMKDVERR ARYVE

UniProtKB: Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein

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Macromolecule #2: Regulatory protein Cgi121

MacromoleculeName: Regulatory protein Cgi121 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 16.963938 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MDPMIIRGIR GARINNEIFN LGLKFQILNA DVVATKKHVL HAINQAKTKK PIAKSFWMEI LVRASGQRQI HEAIKIIGAK DGNVCLICE DEETFRKIYE LIGGEIDDSV LEINEDKERL IREIFKIRGF GNVVERVLEK IALIELKKE

UniProtKB: Regulatory protein Cgi121

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Macromolecule #3: Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesi...

MacromoleculeName: Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Methanocaldococcus jannaschii (archaea)
Molecular weightTheoretical: 36.115688 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MDPMICLGLE GTAEKTGVGI VTSDGEVLFN KTIMYKPPKQ GINPREAADH HAETFPKLIK EAFEVVDKNE IDLIAFSQGP GLGPSLRVT ATVARTLSLT LKKPIIGVNH CIAHIEIGKL TTEAEDPLTL YVSGGNTQVI AYVSKKYRVF GETLDIAVGN C LDQFARYV ...String:
MDPMICLGLE GTAEKTGVGI VTSDGEVLFN KTIMYKPPKQ GINPREAADH HAETFPKLIK EAFEVVDKNE IDLIAFSQGP GLGPSLRVT ATVARTLSLT LKKPIIGVNH CIAHIEIGKL TTEAEDPLTL YVSGGNTQVI AYVSKKYRVF GETLDIAVGN C LDQFARYV NLPHPGGPYI EELARKGKKL VDLPYTVKGM DIAFSGLLTA AMRAYDAGER LEDICYSLQE YAFSMLTEIT ER ALAHTNK GEVMLVGGVA ANNRLREMLK AMCEGQNVDF YVPPKEFCGD NGAMIAWLGL LMHKNGRWMS LDETKIIPNY RTD MVEVNW I

UniProtKB: Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein

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Macromolecule #4: KEOPS complex subunit Pcc1

MacromoleculeName: KEOPS complex subunit Pcc1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 9.410949 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MKAKRVQAKI EIEFPSEDVA KVVYEAVLYE HLSVPYRRSE IDFKLEGKKI ILDIKATDSS ALRGTVNSYL RWIKAAIDVI EV

UniProtKB: KEOPS complex subunit Pcc1

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Macromolecule #5: tRNA

MacromoleculeName: tRNA / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Methanocaldococcus jannaschii (archaea)
Molecular weightTheoretical: 24.413467 KDa
SequenceString:
GGCCCGUAGC UCAGUCUGGC AGAGCGCCUG GCUUUUAACC AGGUGGUCGA GGGUUCAAAU CCCUUCGGGC CCGCCA

GENBANK: GENBANK: L77117.1

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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
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 55.61 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.9 µm / Nominal defocus min: 0.4 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 83710
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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