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- EMDB-28266: Phospholipase C beta 3 (PLCb3) in solution -

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Basic information

Entry
Database: EMDB / ID: EMD-28266
TitlePhospholipase C beta 3 (PLCb3) in solution
Map datafinal sharpened map
Sample
  • Complex: PLCb3 in solution
    • Protein or peptide: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3
  • Ligand: CALCIUM IONCalcium
Function / homology
Function and homology information


phosphoinositide phospholipase C / Fatty Acids bound to GPR40 (FFAR1) regulate insulin secretion / Acetylcholine regulates insulin secretion / phosphatidylinositol metabolic process / PLC beta mediated events / regulation of systemic arterial blood pressure / phosphatidylinositol phospholipase C activity / phospholipase C activity / phosphatidylinositol-mediated signaling / postsynaptic cytosol ...phosphoinositide phospholipase C / Fatty Acids bound to GPR40 (FFAR1) regulate insulin secretion / Acetylcholine regulates insulin secretion / phosphatidylinositol metabolic process / PLC beta mediated events / regulation of systemic arterial blood pressure / phosphatidylinositol phospholipase C activity / phospholipase C activity / phosphatidylinositol-mediated signaling / postsynaptic cytosol / Synthesis of IP3 and IP4 in the cytosol / lipid catabolic process / release of sequestered calcium ion into cytosol / molecular function activator activity / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Ca2+ pathway / G alpha (q) signalling events / molecular adaptor activity / calmodulin binding / cadherin binding / G protein-coupled receptor signaling pathway / calcium ion binding / protein-containing complex / membrane / nucleus / cytosol
Similarity search - Function
Phospholipase C-beta, C-terminal domain / PLC-beta C terminal / PLC-beta, PH domain / Phospholipase C-beta, C-terminal domain superfamily / PH domain / Phosphatidylinositol-4, 5-bisphosphate phosphodiesterase beta / Phosphoinositide-specific phospholipase C, EF-hand-like domain / Phosphoinositide-specific phospholipase C, efhand-like / Phosphoinositide phospholipase C family / Phospholipase C, phosphatidylinositol-specific, Y domain ...Phospholipase C-beta, C-terminal domain / PLC-beta C terminal / PLC-beta, PH domain / Phospholipase C-beta, C-terminal domain superfamily / PH domain / Phosphatidylinositol-4, 5-bisphosphate phosphodiesterase beta / Phosphoinositide-specific phospholipase C, EF-hand-like domain / Phosphoinositide-specific phospholipase C, efhand-like / 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 / Phosphatidylinositol-specific phospholipase X-box domain profile. / Phosphatidylinositol-specific phospholipase C, X domain / Phosphatidylinositol-specific phospholipase C, X domain / Phospholipase C, catalytic domain (part); domain X / PLC-like phosphodiesterase, TIM beta/alpha-barrel domain superfamily / C2 domain / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain profile. / C2 domain superfamily / EF-hand domain pair
Similarity search - Domain/homology
1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsFalzone ME / MacKinnon R
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM142137 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: activates hydrolysis by recruiting and orienting on the membrane surface.
Authors: Maria E Falzone / Roderick MacKinnon /
Abstract: catalyze the hydrolysis of phosphatidylinositol 4, 5-bisphosphate [Formula: see text] into [Formula: see text] [Formula: see text] and [Formula: see text]  [Formula: see text]. [Formula: see text] ... catalyze the hydrolysis of phosphatidylinositol 4, 5-bisphosphate [Formula: see text] into [Formula: see text] [Formula: see text] and [Formula: see text]  [Formula: see text]. [Formula: see text] regulates the activity of many membrane proteins, while and lead to increased intracellular Ca levels and activate protein kinase C, respectively. are regulated by G protein-coupled receptors through direct interaction with [Formula: see text] and [Formula: see text] and are aqueous-soluble enzymes that must bind to the cell membrane to act on their lipid substrate. This study addresses the mechanism by which [Formula: see text] activates 3. We show that 3 functions as a slow Michaelis-Menten enzyme ( [Formula: see text] ) on membrane surfaces. We used membrane partitioning experiments to study the solution-membrane localization equilibrium of 3. Its partition coefficient is such that only a small quantity of 3 exists in the membrane in the absence of [Formula: see text] . When [Formula: see text] is present, equilibrium binding on the membrane surface increases 3 in the membrane, increasing [Formula: see text] in proportion. Atomic structures on membrane vesicle surfaces show that two [Formula: see text] anchor 3 with its catalytic site oriented toward the membrane surface. Taken together, the enzyme kinetic, membrane partitioning, and structural data show that [Formula: see text] activates by increasing its concentration on the membrane surface and orienting its catalytic core to engage [Formula: see text] . This principle of activation explains rapid stimulated catalysis with low background activity, which is essential to the biological processes mediated by [Formula: see text], and .
History
DepositionSep 28, 2022-
Header (metadata) releaseMay 24, 2023-
Map releaseMay 24, 2023-
UpdateMay 24, 2023-
Current statusMay 24, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28266.png

Downloads & links

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Map

FileDownload / File: emd_28266.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfinal sharpened map
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.0204
Minimum - Maximum-0.08873808 - 0.1533337
Average (Standard dev.)7.302812e-05 (±0.0040263142)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: final unsharpened map

Fileemd_28266_additional_1.map
Annotationfinal unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_28266_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_28266_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 : PLCb3 in solution

EntireName: PLCb3 in solution
Components
  • Complex: PLCb3 in solution
    • Protein or peptide: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: PLCb3 in solution

SupramoleculeName: PLCb3 in solution / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 138 KDa

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Macromolecule #1: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3

MacromoleculeName: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: phosphoinositide phospholipase C
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 138.830422 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GPSRATMALQ LEPPTVVETL RRGSKFIKWD EETSSRNLVT LRVDPNGFFL YWTGPNMEVD TLDISSIRDT RTGRYARLPK DPKIREVLG FGGPDARLEE KLMTVVSGPD PVNTVFLNFM AVQDDTAKVW SEELFKLAMN ILAQNASRNT FLRKAYTKLK L QVNQDGRI ...String:
GPSRATMALQ LEPPTVVETL RRGSKFIKWD EETSSRNLVT LRVDPNGFFL YWTGPNMEVD TLDISSIRDT RTGRYARLPK DPKIREVLG FGGPDARLEE KLMTVVSGPD PVNTVFLNFM AVQDDTAKVW SEELFKLAMN ILAQNASRNT FLRKAYTKLK L QVNQDGRI PVKNILKMFS ADKKRVETAL ESCGLKFNRS ESIRPDEFSL EIFERFLNKL CLRPDIDKIL LEIGAKGKPY LT LEQLMDF INQKQRDPRL NEVLYPPLRP SQARLLIEKY EPNQQFLERD QMSMEGFSRY LGGEENGILP LEALDLSTDM TQP LSAYFI NSSHNTYLTA GQLAGTSSVE MYRQALLWGC RCVELDVWKG RPPEEEPFIT HGFTMTTEVP LRDVLEAIAE TAFK TSPYP VILSFENHVD SAKQQAKMAE YCRSIFGDAL LIEPLDKYPL APGVPLPSPQ DLMGRILVKN KKRHRPSAGG PDSAG RKRP LEQSNSALSE SSAATEPSSP QLGSPSSDSC PGLSNGEEVG LEKPSLEPQK SLGDEGLNRG PYVLGPADRE DEEEDE EEE EQTDPKKPTT DEGTASSEVN ATEEMSTLVN YIEPVKFKSF EAARKRNKCF EMSSFVETKA MEQLTKSPME FVEYNKQ QL SRIYPKGTRV DSSNYMPQLF WNVGCQLVAL NFQTLDVAMQ LNAGVFEYNG RSGYLLKPEF MRRPDKSFDP FTEVIVDG I VANALRVKVI SGQFLSDRKV GIYVEVDMFG LPVDTRRKYR TRTSQGNSFN PVWDEEPFDF PKVVLPTLAS LRIAAFEEG GKFVGHRILP VSAIRSGYHY VCLRNEANQP LCLPALLIYT EASDYIPDDH QDYAEALINP IKHVSLMDQR ARQLAALIGE SEAQAGQET CQDTQSQQLG SQPSSNPTPS PLDASPRRPP GPTTSPASTS LSSPGQRDDL IASILSEVAP TPLDELRGHK A LVKLRSRQ ERDLRELRKK HQRKAVTLTR RLLDGLAQAQ AEGRCRLRPG ALGGAADVED TKEGEDEAKR YQEFQNRQVQ SL LELREAQ VDAEAQRRLE HLRQALQRLR EVVLDANTTQ FKRLKEMNER EKKELQKILD RKRHNSISEA KMRDKHKKEA ELT EINRRH ITESVNSIRR LEEAQKQRHD RLVAGQQQVL QQLAEEEPKL LAQLAQECQE QRARLPQEIR RSLLGEMPEG LGDG PLVAC ASNGHAPGSS GHLSGADSES QEENTQL

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / 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

Concentration4.8 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 22 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 3527 / Average exposure time: 2.0 sec. / Average electron dose: 42.87 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1021849 / Details: picked with 2D templates
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 67716
FSC plot (resolution estimation)

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