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- PDB-9d85: Structure of the KEOPS complex (Cgi121/Bud32/Kae1/Pcc1) bound to ... -

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Basic information

Entry
Database: PDB / ID: 9d85
TitleStructure of the KEOPS complex (Cgi121/Bud32/Kae1/Pcc1) bound to tRNA in a distorted tRNA conformation
Components
  • (Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein) x 2
  • KEOPS complex subunit Pcc1
  • Regulatory protein Cgi121
  • tRNA
KeywordsRNA BINDING PROTEIN/RNA / tRNA / modification / t6A / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


N6-L-threonylcarbamoyladenine synthase / tRNA N(6)-L-threonylcarbamoyladenine synthase activity / EKC/KEOPS complex / tRNA threonylcarbamoyladenosine modification / protein serine/threonine/tyrosine kinase activity / metalloendopeptidase activity / non-specific serine/threonine protein kinase / iron ion binding / protein serine kinase activity / protein serine/threonine kinase activity ...N6-L-threonylcarbamoyladenine synthase / tRNA N(6)-L-threonylcarbamoyladenine synthase activity / EKC/KEOPS complex / tRNA threonylcarbamoyladenosine modification / protein serine/threonine/tyrosine kinase activity / metalloendopeptidase activity / non-specific serine/threonine protein kinase / iron ion binding / protein serine kinase activity / protein serine/threonine 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
: / RNA / RNA (> 10) / Regulatory protein Cgi121 / Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein / KEOPS complex subunit Pcc1
Similarity search - Component
Biological speciesMethanocaldococcus jannaschii (archaea)
Pyrococcus furiosus DSM 3638 (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.59 Å
AuthorsOna Chuquimarca, S.M. / Beenstock, J. / Daou, S. / Keszei, A.F.A. / Yin, Z. / Sicheri, F.
Funding support Canada, 1items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 18, 2024Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Dec 18, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 2.0May 14, 2025Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Polymer sequence / Refinement description / Source and taxonomy / Structure summary
Category: atom_site / em_admin ...atom_site / em_admin / em_imaging / em_software / entity / entity_poly / entity_poly_seq / entity_src_gen / ndb_struct_na_base_pair / ndb_struct_na_base_pair_step / pdbx_contact_author / pdbx_poly_seq_scheme / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_close_contact / pdbx_validate_planes / pdbx_validate_rmsd_angle / pdbx_validate_torsion / refine_ls_restr / struct_conf / struct_conn / struct_mon_prot_cis / struct_ref / struct_ref_seq / struct_ref_seq_dif / struct_sheet / struct_sheet_order / struct_sheet_range
Item: _em_admin.last_update / _em_imaging.microscope_model ..._em_admin.last_update / _em_imaging.microscope_model / _entity.formula_weight / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _entity_src_gen.pdbx_end_seq_num / _struct_mon_prot_cis.pdbx_omega_angle / _struct_ref.pdbx_align_begin / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.db_align_beg / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.seq_align_end / _struct_ref_seq_dif.seq_num
Description: Sequence discrepancy
Details: tRNA model had a single nucleotide frame shift due to missing 5' guanosine
Provider: author / Type: Coordinate replacement
Revision 1.1May 14, 2025Data content type: EM metadata
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
Group: Data collection / Data processing ...Data collection / Data processing / Database references / Experimental summary / Other / Source and taxonomy / Structure summary
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
Category: em_admin / em_imaging ...em_admin / em_imaging / em_software / entity / entity_poly / entity_src_gen / struct_ref / struct_ref_seq / struct_ref_seq_dif
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
Item: _em_admin.last_update / _em_imaging.microscope_model ..._em_admin.last_update / _em_imaging.microscope_model / _entity.formula_weight / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _entity_src_gen.pdbx_end_seq_num / _struct_ref.pdbx_align_begin / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.db_align_beg / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.seq_align_end / _struct_ref_seq_dif.seq_num

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein
C: Regulatory protein Cgi121
K: Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein
P: KEOPS complex subunit Pcc1
T: tRNA


Theoretical massNumber of molelcules
Total (without water)111,2075
Polymers111,2075
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 Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein


Mass: 23957.547 Da / Num. of mol.: 1 / Mutation: E152R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methanocaldococcus jannaschii (archaea)
Gene: MJ1130 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q58530
#2: Protein Regulatory protein Cgi121 / Positive regulator of Bud32 kinase activity


Mass: 16963.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: cgi121, MJ0187 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q57646
#3: Protein Probable bifunctional tRNA threonylcarbamoyladenosine biosynthesis protein


Mass: 36115.688 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methanocaldococcus jannaschii (archaea)
Gene: MJ1130 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q58530
#4: Protein KEOPS complex subunit Pcc1


Mass: 9410.949 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: PF2011 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8TZI1
#5: RNA chain tRNA


Mass: 24758.672 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methanocaldococcus jannaschii (archaea)
Production host: in vitro transcription vector pT7-Fluc(deltai) (others)
References: GenBank: 6626255
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

Component
IDNameTypeEntity IDParent-IDSource
1Structure of the KEOPS complex (Cgi121/Bud32/Kae1/Pcc1)COMPLEXall0RECOMBINANT
2Bud32COMPLEX#11RECOMBINANT
3Cgi121COMPLEX#21RECOMBINANT
4Kae1COMPLEX#31RECOMBINANT
5Pcc1COMPLEX1RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Methanocaldococcus jannaschii (archaea)2190
32Methanocaldococcus jannaschii (archaea)2190
43Methanocaldococcus jannaschii (archaea)2190
54Methanocaldococcus jannaschii (archaea)2190
65Pyrococcus furiosus (archaea)2261
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli BL21(DE3) (bacteria)469008
32Escherichia coli BL21(DE3) (bacteria)469008
43Escherichia coli BL21(DE3) (bacteria)469008
54Escherichia coli BL21(DE3) (bacteria)469008
65Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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: 2900 nm / Nominal defocus min: 400 nm
Image recordingElectron dose: 55.61 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83710 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037561
ELECTRON MICROSCOPYf_angle_d0.53610539
ELECTRON MICROSCOPYf_dihedral_angle_d21.1671851
ELECTRON MICROSCOPYf_chiral_restr0.041255
ELECTRON MICROSCOPYf_plane_restr0.0031084

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