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- EMDB-44067: Cryo-EM structure of phospholipase Cepsilon PH-COOH in complex wi... -

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Basic information

Entry
Database: EMDB / ID: EMD-44067
TitleCryo-EM structure of phospholipase Cepsilon PH-COOH in complex with an antigen-binding fragment (focused map)
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: Fab2
Keywordsphospholipase c / antigen-binding fragment / complex / MEMBRANE PROTEIN
Function / homology:
Function and homology information
Biological speciesRattus norvegicus (Norway rat) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 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_44067.png

Downloads & links

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Map

FileDownload / File: emd_44067.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: 0.023
Minimum - Maximum-0.18683435 - 0.266279
Average (Standard dev.)0.00014504613 (±0.005252834)
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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Mask #1

Fileemd_44067_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44067_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_44067_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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: Fab2

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Supramolecule #1: Fab2-rPLCe PH-C

SupramoleculeName: Fab2-rPLCe PH-C / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: fragment phospholipase Cepsilon (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
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 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHSSG VDLGTENLYF QSNADHGTEL IPWYVLSIQA DVHQFLLQGA TVIHYDQDTH LSARCFLQLQ PDNSTLTWMK PPTASPAGAR LKLGVLSNVA EPGKFPSLGN AGVSGLVEGI LDLFSVKAVY MGHPGIDIHT VCVQNKLSSM LLSETGVTLL YGLQTTDNRL ...String:
MHHHHHHSSG VDLGTENLYF QSNADHGTEL IPWYVLSIQA DVHQFLLQGA TVIHYDQDTH LSARCFLQLQ PDNSTLTWMK PPTASPAGAR LKLGVLSNVA EPGKFPSLGN AGVSGLVEGI LDLFSVKAVY MGHPGIDIHT VCVQNKLSSM LLSETGVTLL YGLQTTDNRL LHFVAPKHTA KMLFSGLLEL TTAVRKIRKF PDQRQQWLRK QYVSFYQEDG RYEGPTLAHA VELFGGRRWS TRNPSPGMSA KNAEKPNMQR NNTLGISTTK KKKKMLMRGE SGEVTDDEMA TRKAKMYREC RSRSGSDPQE ANEQEDSEAN VITNPPNPLH SRRAYSLTTA GSPNLATGMS SPISAWSSSS WHGRIKGGMK GFQSFMVSDS NMSFIEFVEL FKSFSIRSRK DLKDIFDIYS VPCNRSASES TPLYTNLTIE ENTNDLQPDL DLLTRNVSDL GLFMKSKQQL SDNQRQISDA IAAASIVTNG TGIESTSLGI FGVGILQLND FLVNCQGEHC TYDEILSIIQ KFEPNISMCH QGLLSFEGFA RFLMDKDNFA SKNDESRENK KDLQLPLSYY YIESSHNTYL TGHQLKGESS VELYSQVLLQ GCRSIELDCW DGDDGMPIIY HGHTLTTKIP FKEVVEAIDR SAFITSDLPI IISIENHCSL PQQRKMAEIF KSVFGEKLVA KFLFETDFSD DPMLPSPDQL RRKVLLKNKK LKAHQTPVDI LKQKAHQLAS MQTQAFTGGN ANPPPASNEE EEDEEDEYDY DYESLSDDNI LEDRPENKSC ADKLQFEYNE EVPKRIKKAD NSSGNKGKVY DMELGEEFYL PQNKKESRQI APELSDLVIY CQAVKFPGLS TLNSSGSGRG KERKSRKSIF GNNPGRMSPG ETASFNRTSG KSSCEGIRQI WEEPPLSPNT SLSAIIRTPK CYHISSLNEN AAKRLCRRYS QKLIQHTACQ LLRTYPAATR IDSSNPNPLM FWLHGIQLVA LNYQTDDLPL HLNAAMFEAN GGCGYVLKPP VLWDKSCPMY QKFSPLERDL DAMDPATYSL TIISGQNVCP SNSTGSPCIE VDVLGMPLDS CHFRTKPIHR NTLNPMWNEQ FLFRVHFEDL VFLRFAVVEN NSSAITAQRI IPLKALKRGY RHLQLRNLHN EILEISSLFI NSRRMEDNPS GSTRPASLMF NTEERKCSQT HKVTVHGVPG PEPFAVFTIN EGTKAKQLLQ QILAVDQDTK LTAADYFLME EKHFISKEKN ECRKQPFQRA VGPEEDIVQI LNSWFPEEGY VGRIVLKPQQ ETLEEKNIVH DDREVILSSE EESFFVQVHD VSPEQPRTVI KAPRVSTAQD VIQQTLCKAK YSYSILNNPN PCDYVLLEEV MKDAPNKKSS TPKSSQRILL DQECVFQAQS KWKGAGKFIL KLKEQVQASR EDKRRGISFA SELKKLTKST KQTRGLTSPP QLVASESVQS KEEKPMGALA SGDTAGYQS

UniProtKB: UNIPROTKB: QP9984

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Macromolecule #2: Fab2

MacromoleculeName: Fab2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: (light chain) MKKNIAFL LASMFVFSIA TNAYASDIQM TQSPSSLSAS VGDRVTITCR ASQSVSSAVA WYQQKPGKAP KLLIYSASSL YSGVPSRFSG SRSGTDFTLT ISSLQPEDFA TYYCQQSSSS LITFGQGTKV EIKRTVAAPS VFIFPPSDSQ LKSGTASVVC ...String:
(light chain) MKKNIAFL LASMFVFSIA TNAYASDIQM TQSPSSLSAS VGDRVTITCR ASQSVSSAVA WYQQKPGKAP KLLIYSASSL YSGVPSRFSG SRSGTDFTLT ISSLQPEDFA TYYCQQSSSS LITFGQGTKV EIKRTVAAPS VFIFPPSDSQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC (heavy chain) MKKNIAF LLASMFVFSI ATNAYAEISE VQLVESGGGL VQPGGSLRLS CAASGFNFSS SSIHWVRQAP GKGLEWVASI SSSSGSTSYA DSVKGRFTIS ADTSKNTAYL QMNSLRAEDT AVYYCARSEY SYQYVYGWWY WNYSAIDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHT

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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: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 147120
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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