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- EMDB-44064: Cryo-EM structure of phospholipase Cepsilon PH-COOH in complex wi... -

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Basic information

Entry
Database: EMDB / ID: EMD-44064
TitleCryo-EM structure of phospholipase Cepsilon PH-COOH in complex with an antigen-binding fragment (composite structure)
Map data
Sample
  • Complex: Fab2-rPLCe PH-C
    • Complex: rPLCe PH-C
      • Protein or peptide: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1
    • Complex: Fab2
      • Protein or peptide: Antigen-binding fragment heavy chain
      • Protein or peptide: Antigen-binding fragment light chain
  • Ligand: CALCIUM ION
Keywordsphospholipase c / antigen-binding fragment / complex / MEMBRANE PROTEIN
Function / homology
Function and homology information


diacylglycerol biosynthetic process / Synthesis of IP3 and IP4 in the cytosol / phosphoinositide phospholipase C / phosphatidylinositol metabolic process / phospholipase C activity / phosphatidylinositol-4,5-bisphosphate phospholipase C activity / glomerulus development / phosphatidylinositol-mediated signaling / lipid catabolic process / positive regulation of lamellipodium assembly ...diacylglycerol biosynthetic process / Synthesis of IP3 and IP4 in the cytosol / phosphoinositide phospholipase C / phosphatidylinositol metabolic process / phospholipase C activity / phosphatidylinositol-4,5-bisphosphate phospholipase C activity / glomerulus development / phosphatidylinositol-mediated signaling / lipid catabolic process / positive regulation of lamellipodium assembly / release of sequestered calcium ion into cytosol / guanyl-nucleotide exchange factor activity / small GTPase binding / epidermal growth factor receptor signaling pathway / lamellipodium / phospholipase C-activating G protein-coupled receptor signaling pathway / Ras protein signal transduction / intracellular signal transduction / G protein-coupled receptor signaling pathway / Golgi membrane / enzyme binding / metal ion binding / plasma membrane / cytosol
Similarity search - Function
1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1 / : / : / Phosphoinositide phospholipase C family / Phospholipase C, phosphatidylinositol-specific, Y domain / Phosphatidylinositol-specific phospholipase C, Y domain / Phosphatidylinositol-specific phospholipase Y-box domain profile. / Phospholipase C, catalytic domain (part); domain Y / Ras association (RalGDS/AF-6) domain / Ras association (RalGDS/AF-6) domain ...1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1 / : / : / Phosphoinositide phospholipase C family / Phospholipase C, phosphatidylinositol-specific, Y domain / Phosphatidylinositol-specific phospholipase C, Y domain / Phosphatidylinositol-specific phospholipase Y-box domain profile. / Phospholipase C, catalytic domain (part); domain Y / Ras association (RalGDS/AF-6) domain / Ras association (RalGDS/AF-6) domain / Phosphatidylinositol-specific phospholipase C, X domain / Phosphatidylinositol-specific phospholipase C, X domain / Phospholipase C, catalytic domain (part); domain X / Phosphatidylinositol-specific phospholipase X-box domain profile. / Ras-associating (RA) domain profile. / Ras-associating (RA) domain / Ras guanine nucleotide exchange factor domain superfamily / Ras guanine-nucleotide exchange factor, catalytic domain superfamily / RasGEF domain / Ras guanine-nucleotide exchange factors catalytic domain profile. / Guanine nucleotide exchange factor for Ras-like small GTPases / Ras guanine-nucleotide exchange factors catalytic domain / PLC-like phosphodiesterase, TIM beta/alpha-barrel domain superfamily / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain / C2 domain profile. / C2 domain superfamily / EF-hand domain pair / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsSamassekou K / Lyon AM
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)1R01HL141076-01 United States
American Heart Association23PRE1011279 United States
CitationJournal: Commun Biol / Year: 2025
Title: Cryo-EM structure of phospholipase Cε defines N-terminal domains and their roles in activity.
Authors: Kadidia Samassekou / Elisabeth E Garland-Kuntz / Vaani Ohri / Isaac J Fisher / Satchal K Erramilli / Kaushik Muralidharan / Livia M Bogdan / Abigail M Gick / Anthony Kossiakoff / Angeline M Lyon /
Abstract: Phospholipase Cε (PLCε) increases intracellular Ca and protein kinase C (PKC) activity in the cardiovascular system in response to stimulation of G protein coupled receptors (GPCRs) and receptor ...Phospholipase Cε (PLCε) increases intracellular Ca and protein kinase C (PKC) activity in the cardiovascular system in response to stimulation of G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). The ability of PLCε to respond to these diverse inputs is due, in part, to multiple, conformationally dynamic regulatory domains. However, this heterogeneity has limited structural studies of the lipase to either individual domains or its catalytic core. Here, we report the 3.9 Å reconstruction of the largest fragment of PLCε to date in complex with an antigen binding fragment (Fab). The structure reveals that PLCε contains a pleckstrin homology (PH) domain and four tandem EF hands, including subfamily-specific insertions and intramolecular interactions with the catalytic core. The structure, together with a model of the holoenzyme, suggest that part of the N-terminus and PH domain may form a surface that supports lipase activity. Functional characterization of this surface confirms it is critical for maximum basal and G protein-stimulated activities. This study provides new insights into the autoinhibited, basal conformation of PLCε and how the N-terminal domains contribute to activity.
History
DepositionMar 12, 2024-
Header (metadata) releaseMar 26, 2025-
Map releaseMar 26, 2025-
UpdateOct 22, 2025-
Current statusOct 22, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44064.png

Downloads & links

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Map

FileDownload / File: emd_44064.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.54 Å/pix.
x 512 pix.
= 275.968 Å		0.54 Å/pix.
x 512 pix.
= 275.968 Å		0.54 Å/pix.
x 512 pix.
= 275.968 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.539 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.34
Minimum - Maximum-36.568793999999997 - 51.275300000000001
Average (Standard dev.)-0.000062923726 (±0.96714824)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 275.968 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_44064_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_44064_half_map_2.map
Projections & Slices
AxesZYX

Projections

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Fab2-rPLCe PH-C

EntireName: Fab2-rPLCe PH-C
Components
  • Complex: Fab2-rPLCe PH-C
    • Complex: rPLCe PH-C
      • Protein or peptide: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1
    • Complex: Fab2
      • Protein or peptide: Antigen-binding fragment heavy chain
      • Protein or peptide: Antigen-binding fragment light chain
  • Ligand: CALCIUM ION

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Supramolecule #1: Fab2-rPLCe PH-C

SupramoleculeName: Fab2-rPLCe PH-C / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: fragment of phospholipase C epsilon (residues 837-2281) in complex with an antigen binding fragment (Fab2)
Molecular weightTheoretical: 489 KDa

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Supramolecule #2: rPLCe PH-C

SupramoleculeName: rPLCe PH-C / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Supramolecule #3: Fab2

SupramoleculeName: Fab2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1

MacromoleculeName: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: phosphoinositide phospholipase C
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 165.314125 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHSSG VDLGTENLYF QSNADHGTEL IPWYVLSIQA DVHQFLLQGA TVIHYDQDTH LSARCFLQLQ PDNSTLTWMK PPTASPAGA RLKLGVLSNV AEPGKFPSLG NAGVSGLVEG ILDLFSVKAV YMGHPGIDIH TVCVQNKLSS MLLSETGVTL L YGLQTTDN ...String:
MHHHHHHSSG VDLGTENLYF QSNADHGTEL IPWYVLSIQA DVHQFLLQGA TVIHYDQDTH LSARCFLQLQ PDNSTLTWMK PPTASPAGA RLKLGVLSNV AEPGKFPSLG NAGVSGLVEG ILDLFSVKAV YMGHPGIDIH TVCVQNKLSS MLLSETGVTL L YGLQTTDN RLLHFVAPKH TAKMLFSGLL ELTTAVRKIR KFPDQRQQWL RKQYVSFYQE DGRYEGPTLA HAVELFGGRR WS TRNPSPG MSAKNAEKPN MQRNNTLGIS TTKKKKKMLM RGESGEVTDD EMATRKAKMY RECRSRSGSD PQEANEQEDS EAN VITNPP NPLHSRRAYS LTTAGSPNLA TGMSSPISAW SSSSWHGRIK GGMKGFQSFM VSDSNMSFIE FVELFKSFSI RSRK DLKDI FDIYSVPCNR SASESTPLYT NLTIEENTND LQPDLDLLTR NVSDLGLFMK SKQQLSDNQR QISDAIAAAS IVTNG TGIE STSLGIFGVG ILQLNDFLVN CQGEHCTYDE ILSIIQKFEP NISMCHQGLL SFEGFARFLM DKDNFASKND ESRENK KDL QLPLSYYYIE SSHNTYLTGH QLKGESSVEL YSQVLLQGCR SIELDCWDGD DGMPIIYHGH TLTTKIPFKE VVEAIDR SA FITSDLPIII SIENHCSLPQ QRKMAEIFKS VFGEKLVAKF LFETDFSDDP MLPSPDQLRR KVLLKNKKLK AHQTPVDI L KQKAHQLASM QTQAFTGGNA NPPPASNEEE EDEEDEYDYD YESLSDDNIL EDRPENKSCA DKLQFEYNEE VPKRIKKAD NSSGNKGKVY DMELGEEFYL PQNKKESRQI APELSDLVIY CQAVKFPGLS TLNSSGSGRG KERKSRKSIF GNNPGRMSPG ETASFNRTS GKSSCEGIRQ IWEEPPLSPN TSLSAIIRTP KCYHISSLNE NAAKRLCRRY SQKLIQHTAC QLLRTYPAAT R IDSSNPNP LMFWLHGIQL VALNYQTDDL PLHLNAAMFE ANGGCGYVLK PPVLWDKSCP MYQKFSPLER DLDAMDPATY SL TIISGQN VCPSNSTGSP CIEVDVLGMP LDSCHFRTKP IHRNTLNPMW NEQFLFRVHF EDLVFLRFAV VENNSSAITA QRI IPLKAL KRGYRHLQLR NLHNEILEIS SLFINSRRME DNPSGSTRPA SLMFNTEERK CSQTHKVTVH GVPGPEPFAV FTIN EGTKA KQLLQQILAV DQDTKLTAAD YFLMEEKHFI SKEKNECRKQ PFQRAVGPEE DIVQILNSWF PEEGYVGRIV LKPQQ ETLE EKNIVHDDRE VILSSEEESF FVQVHDVSPE QPRTVIKAPR VSTAQDVIQQ TLCKAKYSYS ILNNPNPCDY VLLEEV MKD APNKKSSTPK SSQRILLDQE CVFQAQSKWK GAGKFILKLK EQVQASREDK RRGISFASEL KKLTKSTKQT RGLTSPP QL VASESVQSKE EKPMGALASG DTAGYQS

UniProtKB: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1

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Macromolecule #2: Antigen-binding fragment heavy chain

MacromoleculeName: Antigen-binding fragment heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.234482 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MKKNIAFLLA SMFVFSIATN AYAEISEVQL VESGGGLVQP GGSLRLSCAA SGFNFSSSSI HWVRQAPGKG LEWVASISSS SGSTSYADS VKGRFTISAD TSKNTAYLQM NSLRAEDTAV YYCARSEYSY QYVYGWWYWN YSAIDYWGQG TLVTVSSAST K GPSVFPLA ...String:
MKKNIAFLLA SMFVFSIATN AYAEISEVQL VESGGGLVQP GGSLRLSCAA SGFNFSSSSI HWVRQAPGKG LEWVASISSS SGSTSYADS VKGRFTISAD TSKNTAYLQM NSLRAEDTAV YYCARSEYSY QYVYGWWYWN YSAIDYWGQG TLVTVSSAST K GPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CN VNHKPSN TKVDKKVEPK SCDKTHT

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Macromolecule #3: Antigen-binding fragment light chain

MacromoleculeName: Antigen-binding fragment light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.794859 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MKKNIAFLLA SMFVFSIATN AYASDIQMTQ SPSSLSASVG DRVTITCRAS QSVSSAVAWY QQKPGKAPKL LIYSASSLYS GVPSRFSGS RSGTDFTLTI SSLQPEDFAT YYCQQSSSSL ITFGQGTKVE IKRTVAAPSV FIFPPSDSQL KSGTASVVCL L NNFYPREA ...String:
MKKNIAFLLA SMFVFSIATN AYASDIQMTQ SPSSLSASVG DRVTITCRAS QSVSSAVAWY QQKPGKAPKL LIYSASSLYS GVPSRFSGS RSGTDFTLTI SSLQPEDFAT YYCQQSSSSL ITFGQGTKVE IKRTVAAPSV FIFPPSDSQL KSGTASVVCL L NNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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: 8
Component:
ConcentrationFormulaName
20.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMNaClsodium chloride
0.3 mMTCEPtris(2-carboxyethyl)phosphine
0.1 mMEDTAEthylenediaminetetraacetic acid
0.1 mMEGTAegtazic acid
0.005 %DDMn-dodecyl-b-D-maltoside
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 7052 / Average exposure time: 3.2 sec. / Average electron dose: 53.75 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1471782
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 147120
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: PROJECTION MATCHING

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