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- PDB-7lrd: Cryo-EM of the SLFN12-PDE3A complex: Consensus subset model -

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Basic information

Entry
Database: PDB / ID: 7lrd
TitleCryo-EM of the SLFN12-PDE3A complex: Consensus subset model
Components
  • Schlafen family member 12
  • cGMP-inhibited 3',5'-cyclic phosphodiesterase A
KeywordsRNA BINDING PROTEIN / complex / velcrin / molecular glue / DNMDP
Function / homology
Function and homology information


cGMP-inhibited cyclic-nucleotide phosphodiesterase activity / estrogen binding / regulation of ribonuclease activity / positive regulation of oocyte development / regulation of meiotic nuclear division / rRNA catabolic process / cellular response to cGMP / oocyte maturation / positive regulation of vascular permeability / negative regulation of vascular permeability ...cGMP-inhibited cyclic-nucleotide phosphodiesterase activity / estrogen binding / regulation of ribonuclease activity / positive regulation of oocyte development / regulation of meiotic nuclear division / rRNA catabolic process / cellular response to cGMP / oocyte maturation / positive regulation of vascular permeability / negative regulation of vascular permeability / negative regulation of cAMP-mediated signaling / 3',5'-cyclic-nucleotide phosphodiesterase / cGMP-mediated signaling / nuclear estrogen receptor activity / 3',5'-cyclic-GMP phosphodiesterase activity / cAMP-mediated signaling / 3',5'-cyclic-nucleotide phosphodiesterase activity / 3',5'-cyclic-AMP phosphodiesterase activity / RNA nuclease activity / cellular response to transforming growth factor beta stimulus / apoptotic signaling pathway / lipid metabolic process / ribosome binding / G alpha (s) signalling events / Hydrolases; Acting on ester bonds / response to xenobiotic stimulus / G protein-coupled receptor signaling pathway / negative regulation of apoptotic process / membrane / metal ion binding / nucleus / cytosol
Similarity search - Function
: / Schlafen, GTPase-like domain / Orthopoxvirus B3 protein / Poxviridae B3 protein / Schlafen family / Schlafen, AlbA_2 domain / Schlafen, AlbA_2 domain superfamily / Schlafen, AlbA_2 / 3'5'-cyclic nucleotide phosphodiesterase, catalytic domain / 3'5'-cyclic nucleotide phosphodiesterase, conserved site ...: / Schlafen, GTPase-like domain / Orthopoxvirus B3 protein / Poxviridae B3 protein / Schlafen family / Schlafen, AlbA_2 domain / Schlafen, AlbA_2 domain superfamily / Schlafen, AlbA_2 / 3'5'-cyclic nucleotide phosphodiesterase, catalytic domain / 3'5'-cyclic nucleotide phosphodiesterase, conserved site / 3'5'-cyclic nucleotide phosphodiesterase, catalytic domain superfamily / 3'5'-cyclic nucleotide phosphodiesterase / 3'5'-cyclic nucleotide phosphodiesterase domain signature. / 3'5'-cyclic nucleotide phosphodiesterase domain profile. / Metal dependent phosphohydrolases with conserved 'HD' motif. / HD/PDEase domain
Similarity search - Domain/homology
: / Chem-X5M / cGMP-inhibited 3',5'-cyclic phosphodiesterase 3A / Ribonuclease SLFN12
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsFuller, J.R. / Garvie, C.W. / Lemke, C.T.
CitationJournal: Nat Commun / Year: 2021
Title: Structure of PDE3A-SLFN12 complex reveals requirements for activation of SLFN12 RNase.
Authors: Colin W Garvie / Xiaoyun Wu / Malvina Papanastasiou / Sooncheol Lee / James Fuller / Gavin R Schnitzler / Steven W Horner / Andrew Baker / Terry Zhang / James P Mullahoo / Lindsay Westlake / ...Authors: Colin W Garvie / Xiaoyun Wu / Malvina Papanastasiou / Sooncheol Lee / James Fuller / Gavin R Schnitzler / Steven W Horner / Andrew Baker / Terry Zhang / James P Mullahoo / Lindsay Westlake / Stephanie H Hoyt / Marcus Toetzl / Matthew J Ranaghan / Luc de Waal / Joseph McGaunn / Bethany Kaplan / Federica Piccioni / Xiaoping Yang / Martin Lange / Adrian Tersteegen / Donald Raymond / Timothy A Lewis / Steven A Carr / Andrew D Cherniack / Christopher T Lemke / Matthew Meyerson / Heidi Greulich /
Abstract: DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated ...DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.
History
DepositionFeb 16, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 9, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 28, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 6, 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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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-23495
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Schlafen family member 12
B: cGMP-inhibited 3',5'-cyclic phosphodiesterase A
C: Schlafen family member 12
D: cGMP-inhibited 3',5'-cyclic phosphodiesterase A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)250,37312
Polymers249,4734
Non-polymers9008
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 4 molecules ACBD

#1: Protein Schlafen family member 12


Mass: 67383.734 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLFN12 / Production host: unidentified baculovirus / References: UniProt: Q8IYM2
#2: Protein cGMP-inhibited 3',5'-cyclic phosphodiesterase A / Cyclic GMP-inhibited phosphodiesterase A / CGI-PDE A


Mass: 57352.996 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PDE3A / Production host: unidentified baculovirus
References: UniProt: Q14432, 3',5'-cyclic-nucleotide phosphodiesterase

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Non-polymers , 4 types, 8 molecules

#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-X5M / (4~{R})-3-[4-(diethylamino)-3-[oxidanyl(oxidanylidene)-$l^{4}-azanyl]phenyl]-4-methyl-4,5-dihydro-1~{H}-pyridazin-6-one


Mass: 305.352 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C15H21N4O3 / Feature type: SUBJECT OF INVESTIGATION

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Details

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: SLFN12-PDE3A complex, consensus subset model / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Details: 0.0038% NP-40s detergent (CAS 9016-45-9) added immediately prior to plunge freezing
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
21 mMmagnesium chlorideMgCl21
320 mMHEPESC8H18N2O4S1
40.5 mMTCEPC9H15O6P1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K / Details: 4.5 second blot time

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3163
Details: Exposures were collected in super-resolution mode, as movies fractionated over 40 frames
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
2EPU2.5image acquisition
4CTFFIND4.1.13CTF correction
9cisTEM1.0.0-betainitial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13UCSF Chimera1.13model refinement
14Coot0.8.9model refinement
CTF correctionDetails: CTF correction was applied throughout the entire processing workflow, beginning with particle picking
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2160426
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47632 / Algorithm: FOURIER SPACE / Details: Relion 3D refinement / Symmetry type: POINT
Atomic model buildingSpace: REAL
Details: The model was generated by rigid body fitting of the SLFN12 and PDE3A bodies (from multi-body refinement and deposited separately) into this map using UCSF Chimera and Coot, followed by ...Details: The model was generated by rigid body fitting of the SLFN12 and PDE3A bodies (from multi-body refinement and deposited separately) into this map using UCSF Chimera and Coot, followed by local refinement in Coot of the linkers between the bodies to ameliorate backbone geometry.
Atomic model buildingPDB-ID: 5YD0

5yd0


Accession code: 5YD0 / Source name: PDB / Type: experimental model

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