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- PDB-8a11: Cryo-EM structure of the Human SHMT1-RNA complex -

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Basic information

Entry
Database: PDB / ID: 8a11
TitleCryo-EM structure of the Human SHMT1-RNA complex
ComponentsSerine hydroxymethyltransferase, cytosolic
KeywordsTRANSFERASE / Riboregulation / Metabolism / 1 carbon metablism / Moonlighting protein / RNA BINDING PROTEIN
Function / homology
Function and homology information


cellular response to tetrahydrofolate / Carnitine synthesis / carnitine biosynthetic process / purine nucleobase biosynthetic process / serine binding / aldehyde-lyase activity / L-serine catabolic process / L-serine metabolic process / glycine metabolic process / glycine hydroxymethyltransferase ...cellular response to tetrahydrofolate / Carnitine synthesis / carnitine biosynthetic process / purine nucleobase biosynthetic process / serine binding / aldehyde-lyase activity / L-serine catabolic process / L-serine metabolic process / glycine metabolic process / glycine hydroxymethyltransferase / glycine hydroxymethyltransferase activity / glycine biosynthetic process from serine / Metabolism of folate and pterines / tetrahydrofolate metabolic process / tetrahydrofolate interconversion / dTMP biosynthetic process / small molecule binding / folic acid metabolic process / mRNA regulatory element binding translation repressor activity / cellular response to leukemia inhibitory factor / mRNA 5'-UTR binding / pyridoxal phosphate binding / protein homotetramerization / negative regulation of translation / protein homodimerization activity / mitochondrion / extracellular exosome / nucleoplasm / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Serine hydroxymethyltransferase, pyridoxal phosphate binding site / Serine hydroxymethyltransferase pyridoxal-phosphate attachment site. / : / Serine hydroxymethyltransferase / Serine hydroxymethyltransferase-like domain / Serine hydroxymethyltransferase / Pyridoxal phosphate-dependent transferase, small domain / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase
Similarity search - Domain/homology
PYRIDOXAL-5'-PHOSPHATE / Serine hydroxymethyltransferase, cytosolic
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsSpizzichino, S. / Marabelli, C. / Bharadwaj, A. / Jakobi, A.J. / Chaves-Sanjuan, A. / Giardina, G. / Bolognesi, M. / Cutruzzola, F.
Funding support Italy, 2items
OrganizationGrant numberCountry
Italian Association for Cancer ResearchAIRC IG-23125 Italy
Other private
CitationJournal: Mol Cell / Year: 2024
Title: Structure-based mechanism of riboregulation of the metabolic enzyme SHMT1.
Authors: Sharon Spizzichino / Federica Di Fonzo / Chiara Marabelli / Angela Tramonti / Antonio Chaves-Sanjuan / Alessia Parroni / Giovanna Boumis / Francesca Romana Liberati / Alessio Paone / Linda ...Authors: Sharon Spizzichino / Federica Di Fonzo / Chiara Marabelli / Angela Tramonti / Antonio Chaves-Sanjuan / Alessia Parroni / Giovanna Boumis / Francesca Romana Liberati / Alessio Paone / Linda Celeste Montemiglio / Matteo Ardini / Arjen J Jakobi / Alok Bharadwaj / Paolo Swuec / Gian Gaetano Tartaglia / Alessandro Paiardini / Roberto Contestabile / Antonello Mai / Dante Rotili / Francesco Fiorentino / Alberto Macone / Alessandra Giorgi / Giancarlo Tria / Serena Rinaldo / Martino Bolognesi / Giorgio Giardina / Francesca Cutruzzolà /
Abstract: RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on ...RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on structural grounds. Here, we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We have determined the cryoelectron microscopy (cryo-EM) structure of human SHMT1 in its free- and RNA-bound states, and we show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme's reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in evolution of eukaryotic SHMT1 and in compartmentalization of one-carbon metabolism. Our findings provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.
History
DepositionMay 30, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release
Revision 2.0May 8, 2024Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Database references / Derived calculations / Non-polymer description / Polymer sequence / Refinement description / Source and taxonomy / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_entity_assembly / em_entity_assembly_molwt / entity / entity_poly / entity_poly_seq / ndb_struct_na_base_pair / pdbx_contact_author / pdbx_entity_instance_feature / pdbx_entity_nonpoly / pdbx_entity_src_syn / pdbx_entry_details / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop / pdbx_struct_mod_residue / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_residues / pdbx_validate_torsion / struct_asym / struct_conf / struct_conn / struct_conn_type / struct_mon_prot_cis / struct_ref / struct_ref_seq / struct_sheet / struct_sheet_order / struct_sheet_range
Item: _chem_comp.formula / _chem_comp.formula_weight ..._chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.pdbx_synonyms / _chem_comp.type / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.chain_id / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_entity_assembly.entity_id_list / _em_entity_assembly_molwt.units / _em_entity_assembly_molwt.value / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_mutation / _entity.pdbx_number_of_molecules / _entity.type / _pdbx_entry_details.has_ligand_of_interest / _pdbx_struct_assembly.oligomeric_count / _pdbx_struct_assembly.oligomeric_details / _pdbx_struct_assembly_gen.asym_id_list / _struct_mon_prot_cis.pdbx_omega_angle
Description: Model completeness
Details: As requested from the reviewers we removed the previously modelled RNA and the PLP molecule from the active site when it's not visible.
Provider: author / Type: Coordinate replacement
Revision 2.1Jul 24, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Serine hydroxymethyltransferase, cytosolic
B: Serine hydroxymethyltransferase, cytosolic
C: Serine hydroxymethyltransferase, cytosolic
D: Serine hydroxymethyltransferase, cytosolic
hetero molecules


Theoretical massNumber of molelcules
Total (without water)214,4817
Polymers213,7394
Non-polymers7413
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, native gel electrophoresis, gel filtration, only for the protein tetramer
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Serine hydroxymethyltransferase, cytosolic / SHMT / Glycine hydroxymethyltransferase / Serine methylase


Mass: 53434.797 Da / Num. of mol.: 4 / Mutation: Chain B has not PLP bond to the active site
Source method: isolated from a genetically manipulated source
Details: first 3 N-ter residues come from removal of His-tag. Sequence numbering starts from first M residue. K257 is covalently bound to PLP forming an internal aldimine (LLP)
Source: (gene. exp.) Homo sapiens (human) / Gene: SHMT1 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P34896, glycine hydroxymethyltransferase
#2: Chemical ChemComp-PLP / PYRIDOXAL-5'-PHOSPHATE / VITAMIN B6 Phosphate


Mass: 247.142 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H10NO6P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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

ComponentName: human SHMT1 in complex with RNA (SHMT2 5' UTR 1-50) / Type: COMPLEX / Details: SHMT1= homotetramer of 53kDa subunits / Entity ID: #1 / Source: RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.21 MDaNO
2153 kDa/nmNO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.2
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
220 mMHepesC8H18N2O4S1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blotted for 4 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 120000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5450

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPU2.8image acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
8Coot0.9.6model fitting
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
20PHENIX1-17-1-3660model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4900000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94430 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 132 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 1BJ4

1bj4


Pdb chain-ID: A / Accession code: 1BJ4 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 133.31 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001514943
ELECTRON MICROSCOPYf_angle_d0.410420257
ELECTRON MICROSCOPYf_chiral_restr0.03532247
ELECTRON MICROSCOPYf_plane_restr0.0034488
ELECTRON MICROSCOPYf_dihedral_angle_d9.03662147

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