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- EMDB-23495: Cryo-EM of the SLFN12-PDE3A complex: Consensus subset model -

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Basic information

Entry
Database: EMDB / ID: EMD-23495
TitleCryo-EM of the SLFN12-PDE3A complex: Consensus subset model
Map dataPrimary Relion refinement output
Sample
  • Complex: SLFN12-PDE3A complex, consensus subset model
    • Protein or peptide: Schlafen family member 12
    • Protein or peptide: cGMP-inhibited 3',5'-cyclic phosphodiesterase A
  • Ligand: ZINC ION
  • Ligand: MANGANESE (II) ION
  • Ligand: MAGNESIUM ION
  • Ligand: (4~{R})-3-[4-(diethylamino)-3-[oxidanyl(oxidanylidene)-$l^{4}-azanyl]phenyl]-4-methyl-4,5-dihydro-1~{H}-pyridazin-6-one
Keywordscomplex / velcrin / molecular glue / DNMDP / RNA BINDING PROTEIN
Function / homology
Function and homology information


catalytic activity, acting on a rRNA / 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 ...catalytic activity, acting on a rRNA / 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
cGMP-inhibited 3',5'-cyclic phosphodiesterase 3A / Ribonuclease SLFN12
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsFuller JR / Garvie CW / Lemke CT
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, 2021-
Header (metadata) releaseJun 9, 2021-
Map releaseJun 9, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7lrd
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23495.png

Downloads & links

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Map

FileDownload / File: emd_23495.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary Relion refinement output
Voxel sizeX=Y=Z: 1.063 Å
Density
Contour LevelBy AUTHOR: 0.0175 / Movie #1: 0.0175
Minimum - Maximum-0.019905115 - 0.057889644
Average (Standard dev.)-0.000000023891268 (±0.0017169513)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 344.412 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0631.0631.063
M x/y/z324324324
origin x/y/z0.0000.0000.000
length x/y/z344.412344.412344.412
α/β/γ90.00090.00090.000
start NX/NY/NZ727265
NX/NY/NZ157157169
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS324324324
D min/max/mean-0.0200.058-0.000

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Supplemental data

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Mask #1

Fileemd_23495_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram (log scale)

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Additional map: Primary map, sharpened by an automatically-determined B-factor (-98.5043)...

Fileemd_23495_additional_1.map
AnnotationPrimary map, sharpened by an automatically-determined B-factor (-98.5043) and filtered to local resolution, using the Relion local resolution implementation.
Projections & Slices
AxesZYX

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Half map: Half-map 2 from Relion refinement

Fileemd_23495_half_map_1.map
AnnotationHalf-map 2 from Relion refinement
Projections & Slices
AxesZYX

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Histogram (log scale)

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Half map: Half-map 1 from Relion refinement

Fileemd_23495_half_map_2.map
AnnotationHalf-map 1 from Relion refinement
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Sample components

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Entire : SLFN12-PDE3A complex, consensus subset model

EntireName: SLFN12-PDE3A complex, consensus subset model
Components
  • Complex: SLFN12-PDE3A complex, consensus subset model
    • Protein or peptide: Schlafen family member 12
    • Protein or peptide: cGMP-inhibited 3',5'-cyclic phosphodiesterase A
  • Ligand: ZINC ION
  • Ligand: MANGANESE (II) ION
  • Ligand: MAGNESIUM ION
  • Ligand: (4~{R})-3-[4-(diethylamino)-3-[oxidanyl(oxidanylidene)-$l^{4}-azanyl]phenyl]-4-methyl-4,5-dihydro-1~{H}-pyridazin-6-one

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Supramolecule #1: SLFN12-PDE3A complex, consensus subset model

SupramoleculeName: SLFN12-PDE3A complex, consensus subset model / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Schlafen family member 12

MacromoleculeName: Schlafen family member 12 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 67.383734 KDa
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: GGGGSMNISV DLETNYAELV LDVGRVTLGE NSRKKMKDCK LRKKQNESVS RAMCALLNSG GGVIKAEIEN EDYSYTKDGI GLDLENSFS NILLFVPEYL DFMQNGNYFL IFVKSWSLNT SGLRITTLSS NLYKRDITSA KVMNATAALE FLKDMKKTRG R LYLRPELL ...String:
GGGGSMNISV DLETNYAELV LDVGRVTLGE NSRKKMKDCK LRKKQNESVS RAMCALLNSG GGVIKAEIEN EDYSYTKDGI GLDLENSFS NILLFVPEYL DFMQNGNYFL IFVKSWSLNT SGLRITTLSS NLYKRDITSA KVMNATAALE FLKDMKKTRG R LYLRPELL AKRPCVDIQE ENNMKALAGV FFDRTELDRK EKLTFTESTH VEIKNFSTEK LLQRIKEILP QYVSAFANTD GG YLFIGLN EDKEIIGFKA EMSDLDDLER EIEKSIRKMP VHHFCMEKKK INYSCKFLGV YDKGSLCGYV CALRVERFCC AVF AKEPDS WHVKDNRVMQ LTRKEWIQFM VEAEPKFSSS YEEVISQINT SLPAPHSWPL LEWQRQRHHC PGLSGRITYT PENL CRKLF LQHEGLKQLI CEEMDSVRKG SLIFSRSWSV DLGLQENHKV LCDALLISQD SPPVLYTFHM VQDEEFKGYS TQTAL TLKQ KLAKIGGYTK KVCVMTKIFY LSPEGMTSCQ YDLRSQVIYP ESYYFTRRKY LLKALFKALK RLKSLRDQFS FAENLY QII GIDCFQKNDK KMFKSCRRLT

UniProtKB: Ribonuclease SLFN12

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Macromolecule #2: cGMP-inhibited 3',5'-cyclic phosphodiesterase A

MacromoleculeName: cGMP-inhibited 3',5'-cyclic phosphodiesterase A / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: 3',5'-cyclic-nucleotide phosphodiesterase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 57.352996 KDa
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: GEDETECLRE PLRKASACST YAPETMMFLD KPILAPEPLV MDNLDSIMEQ LNTWNFPIFD LVENIGRKCG RILSQVSYRL FEDMGLFEA FKIPIREFMN YFHALEIGYR DIPYHNRIHA TDVLHAVWYL TTQPIPGLST VINDHGSTSD SDSDSGFTHG H MGYVFSKT ...String:
GEDETECLRE PLRKASACST YAPETMMFLD KPILAPEPLV MDNLDSIMEQ LNTWNFPIFD LVENIGRKCG RILSQVSYRL FEDMGLFEA FKIPIREFMN YFHALEIGYR DIPYHNRIHA TDVLHAVWYL TTQPIPGLST VINDHGSTSD SDSDSGFTHG H MGYVFSKT YNVTDDKYGC LSGNIPALEL MALYVAAAMH DYDHPGRTNA FLVATSAPQA VLYNDRSVLE NHHAAAAWNL FM SRPEYNF LINLDHVEFK HFRFLVIEAI LATDLKKHFD FVAKFNGKVN DDVGIDWTNE NDRLLVCQMC IKLADINGPA KCK ELHLQW TDGIVNEFYE QGDEEASLGL PISPFMDRSA PQLANLQESF ISHIVGPLCN SYDSAGLMPG KWVEDSDESG DTDD PEEEE EEAPAPNEEE TCENNESPKK KTFKRRKIYC QITQHLLQNH KMWKKVIEEE QRLAGIENQS LDQTPQSHSS EQIQA IKEE EEEKGKPRGE EIPTQKPDQ

UniProtKB: cGMP-inhibited 3',5'-cyclic phosphodiesterase 3A

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #4: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: (4~{R})-3-[4-(diethylamino)-3-[oxidanyl(oxidanylidene)-$l^{4}-aza...

MacromoleculeName: (4~{R})-3-[4-(diethylamino)-3-[oxidanyl(oxidanylidene)-$l^{4}-azanyl]phenyl]-4-methyl-4,5-dihydro-1~{H}-pyridazin-6-one
type: ligand / ID: 6 / Number of copies: 2 / Formula: X5M
Molecular weightTheoretical: 305.352 Da
Chemical component information

ChemComp-X5M:
(4~{R})-3-[4-(diethylamino)-3-[oxidanyl(oxidanylidene)-$l^{4}-azanyl]phenyl]-4-methyl-4,5-dihydro-1~{H}-pyridazin-6-one

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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

Concentration0.6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
1.0 mMMgCl2magnesium chloride
20.0 mMC8H18N2O4SHEPES
0.5 mMC9H15O6PTCEP

Details: 0.0038% NP-40s detergent (CAS 9016-45-9) added immediately prior to plunge freezing
GridModel: C-flat / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV / Details: 4.5 second blot time.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 3163 / Average electron dose: 50.0 e/Å2
Details: Exposures were collected in super-resolution mode, as movies fractionated over 40 frames
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2160426
Startup modelType of model: OTHER
Details: The initial startup model was calculated de novo from the data using the method implemented in cisTEM v1.0.0-beta
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM (ver. 1.0.0-beta)
Final 3D classificationNumber classes: 9 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Details: Relion 3D refinement / Number images used: 47632
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5yd0


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsThe 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.
RefinementSpace: REAL
Output model

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

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