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- PDB-6z3r: Structure of SMG1-8-9 kinase complex bound to UPF1-LSQ -

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Basic information

Entry
Database: PDB / ID: 6z3r
TitleStructure of SMG1-8-9 kinase complex bound to UPF1-LSQ
Components
  • Protein SMG8
  • Protein SMG9
  • Regulator of nonsense transcripts 1
  • Serine/threonine-protein kinase SMG1,Serine/threonine-protein kinase SMG1,SMG1,Serine/threonine-protein kinase SMG1,Serine/threonine-protein kinase SMG1,Serine/threonine-protein kinase SMG1
KeywordsTRANSFERASE / Cryo-EM / Structural Biology / Nonsense-mediated mRNA decay / RNA quality control / PIKK / NMD / Substrate / Phosphorylation
Function / homology
Function and homology information


double-stranded DNA helicase activity / supraspliceosomal complex / positive regulation of mRNA cis splicing, via spliceosome / exon-exon junction complex / telomere maintenance via semi-conservative replication / positive regulation of mRNA catabolic process / cell cycle phase transition / diacylglycerol-dependent serine/threonine kinase activity / regulation of translational termination / chromatoid body ...double-stranded DNA helicase activity / supraspliceosomal complex / positive regulation of mRNA cis splicing, via spliceosome / exon-exon junction complex / telomere maintenance via semi-conservative replication / positive regulation of mRNA catabolic process / cell cycle phase transition / diacylglycerol-dependent serine/threonine kinase activity / regulation of translational termination / chromatoid body / histone mRNA catabolic process / eye development / 3'-UTR-mediated mRNA destabilization / regulation of protein kinase activity / regulation of telomere maintenance / nuclear-transcribed mRNA catabolic process / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / DNA duplex unwinding / telomeric DNA binding / phosphatidylinositol phosphate biosynthetic process / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / cellular response to interleukin-1 / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / mRNA export from nucleus / helicase activity / P-body / brain development / heart development / peptidyl-serine phosphorylation / DNA helicase / in utero embryonic development / cellular response to lipopolysaccharide / DNA replication / protein autophosphorylation / chromosome, telomeric region / RNA helicase activity / non-specific serine/threonine protein kinase / protein kinase activity / RNA helicase / protein serine kinase activity / DNA repair / protein serine/threonine kinase activity / DNA damage response / chromatin binding / chromatin / protein-containing complex binding / negative regulation of apoptotic process / perinuclear region of cytoplasm / ATP hydrolysis activity / RNA binding / zinc ion binding / nucleoplasm / ATP binding / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Upf1 cysteine-histidine-rich (CH-rich) domain profile. / RNA helicase UPF1, 1B domain / RNA helicase (UPF2 interacting domain) / RNA helicase UPF1, 1B domain / RNA helicase UPF1, Cys/His rich zinc-binding domain / Nonsense-mediated mRNA decay factor SMG8/SMG9 / Smg8_Smg9 / Nonsense-mediated mRNA decay factor SMG9 / Serine/threonine-protein kinase SMG1 / Serine/threonine-protein kinase SMG1, N-terminal ...Upf1 cysteine-histidine-rich (CH-rich) domain profile. / RNA helicase UPF1, 1B domain / RNA helicase (UPF2 interacting domain) / RNA helicase UPF1, 1B domain / RNA helicase UPF1, Cys/His rich zinc-binding domain / Nonsense-mediated mRNA decay factor SMG8/SMG9 / Smg8_Smg9 / Nonsense-mediated mRNA decay factor SMG9 / Serine/threonine-protein kinase SMG1 / Serine/threonine-protein kinase SMG1, N-terminal / SMG1, PIKK catalytic domain / Serine/threonine-protein kinase smg-1 / Serine/threonine-protein kinase SMG1 N-terminal / : / DNA2/NAM7 helicase, helicase domain / DNA2/NAM7-like helicase / AAA domain / Rapamycin binding domain / FATC domain / Helicase/UvrB, N-terminal / Type III restriction enzyme, res subunit / FATC / FATC domain / PIK-related kinase / FAT domain profile. / FATC domain profile. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / Armadillo-like helical / DNA2/NAM7 helicase-like, C-terminal / AAA domain / Armadillo-type fold / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / ADENOSINE-5'-TRIPHOSPHATE / INOSITOL HEXAKISPHOSPHATE / Nonsense-mediated mRNA decay factor SMG8 / Regulator of nonsense transcripts 1 / Serine/threonine-protein kinase SMG1 / Nonsense-mediated mRNA decay factor SMG9
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsLanger, L.M. / Gat, Y. / Conti, E.
CitationJournal: Elife / Year: 2020
Title: Structure of substrate-bound SMG1-8-9 kinase complex reveals molecular basis for phosphorylation specificity.
Authors: Lukas M Langer / Yair Gat / Fabien Bonneau / Elena Conti /
Abstract: PI3K-related kinases (PIKKs) are large Serine/Threonine (Ser/Thr)-protein kinases central to the regulation of many fundamental cellular processes. PIKK family member SMG1 orchestrates progression of ...PI3K-related kinases (PIKKs) are large Serine/Threonine (Ser/Thr)-protein kinases central to the regulation of many fundamental cellular processes. PIKK family member SMG1 orchestrates progression of an RNA quality control pathway, termed nonsense-mediated mRNA decay (NMD), by phosphorylating the NMD factor UPF1. Phosphorylation of UPF1 occurs in its unstructured N- and C-terminal regions at Serine/Threonine-Glutamine (SQ) motifs. How SMG1 and other PIKKs specifically recognize SQ motifs has remained unclear. Here, we present a cryo-electron microscopy (cryo-EM) reconstruction of a human SMG1-8-9 kinase complex bound to a UPF1 phosphorylation site at an overall resolution of 2.9 Å. This structure provides the first snapshot of a human PIKK with a substrate-bound active site. Together with biochemical assays, it rationalizes how SMG1 and perhaps other PIKKs specifically phosphorylate Ser/Thr-containing motifs with a glutamine residue at position +1 and a hydrophobic residue at position -1, thus elucidating the molecular basis for phosphorylation site recognition.
History
DepositionMay 21, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 24, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 7, 2020Group: Structure summary / Category: chem_comp / Item: _chem_comp.pdbx_synonyms
Revision 1.2May 22, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Serine/threonine-protein kinase SMG1,Serine/threonine-protein kinase SMG1,SMG1,Serine/threonine-protein kinase SMG1,Serine/threonine-protein kinase SMG1,Serine/threonine-protein kinase SMG1
B: Protein SMG8
C: Protein SMG9
E: Regulator of nonsense transcripts 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)550,0628
Polymers548,3644
Non-polymers1,6984
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13340 Å2
ΔGint-91 kcal/mol
Surface area117700 Å2
MethodPISA

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein Serine/threonine-protein kinase SMG1,Serine/threonine-protein kinase SMG1,SMG1,Serine/threonine-protein kinase SMG1,Serine/threonine-protein kinase SMG1,Serine/threonine-protein kinase SMG1 / hSMG-1 / 61E3.4 / Lambda/iota protein kinase C-interacting protein / Lambda-interacting protein


Mass: 379584.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SMG1, ATX, KIAA0421, LIP / Cell line (production host): HEK293T / Production host: Homo sapiens (human)
References: UniProt: Q96Q15, non-specific serine/threonine protein kinase
#2: Protein Protein SMG8 / Amplified in breast cancer gene 2 protein / Protein smg-8 homolog


Mass: 109825.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SMG8, ABC2, C17orf71 / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q8ND04
#3: Protein Protein SMG9 / Protein SMG-9


Mass: 57717.473 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SMG9, C19orf61 / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q9H0W8

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Protein/peptide , 1 types, 1 molecules E

#4: Protein/peptide Regulator of nonsense transcripts 1 / ATP-dependent helicase RENT1 / Nonsense mRNA reducing factor 1 / NORF1 / Up-frameshift suppressor 1 ...ATP-dependent helicase RENT1 / Nonsense mRNA reducing factor 1 / NORF1 / Up-frameshift suppressor 1 homolog / hUpf1


Mass: 1236.286 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
References: UniProt: Q92900, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement

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Non-polymers , 4 types, 4 molecules

#5: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#6: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE / Phytic acid


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6
#7: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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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
1SMG1-8-9 bound to AMPPNP and UPF1-LSQCOMPLEX#1-#40MULTIPLE SOURCES
2SMG1-8-9COMPLEX#1-#31RECOMBINANT
3UPF1-LSQCOMPLEX#41RECOMBINANT
Molecular weightValue: 0.577 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23synthetic construct (others)32630
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 5.5 sec. / Electron dose: 68.75 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6293
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
9PHENIX1.17model refinement
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4368586
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 481754 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 6SYT

6syt


Accession code: 6SYT / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00436594
ELECTRON MICROSCOPYf_angle_d0.74665570
ELECTRON MICROSCOPYf_dihedral_angle_d7.82314930
ELECTRON MICROSCOPYf_chiral_restr0.0793254
ELECTRON MICROSCOPYf_plane_restr0.0045712

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