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- PDB-8emx: Phospholipase C beta 3 (PLCb3) in complex with Gbg on lipid nanodiscs -

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Basic information

Entry
Database: PDB / ID: 8emx
TitlePhospholipase C beta 3 (PLCb3) in complex with Gbg on lipid nanodiscs
Components
  • 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3
  • Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
  • Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
KeywordsHYDROLASE / PIP2 degradation / IP3 production / DAG production / G protein signaling
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 / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / GPER1 signaling / G-protein beta-subunit binding / heterotrimeric G-protein complex / Inactivation, recovery and regulation of the phototransduction cascade / extracellular vesicle / G alpha (12/13) signalling events / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / GTPase binding / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / Ras protein signal transduction / cell population proliferation / Extra-nuclear estrogen signaling / molecular adaptor activity / calmodulin binding / cadherin binding / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / synapse / calcium ion binding / protein-containing complex binding / signal transduction / protein-containing complex / extracellular exosome / membrane / nucleus / plasma membrane / cytosol / cytoplasm
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 / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / EF-hand domain pair / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsFalzone, M.E. / MacKinnon, R.
Funding support United States, 2items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 24, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
C: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
D: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)229,3656
Polymers229,3255
Non-polymers401
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, the complex was present in the structure.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3 / Phosphoinositide phospholipase C-beta-3 / Phospholipase C-beta-3 / PLC-beta-3


Mass: 139055.625 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PLCB3 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q01970, phosphoinositide phospholipase C
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37416.930 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P62873
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7717.917 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P59768
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Phospholipase C beta 3 (PLCb3) in complex with two copies of Gbg on lipid nanodiscs.COMPLEX#1-#30RECOMBINANT
2Phospholipase C beta 3 (PLCb3)COMPLEX#11RECOMBINANT
3G protein beta subunit (Gb1)COMPLEX#21RECOMBINANT
4G protein gamma subunit 2 (Gg2)COMPLEX#31RECOMBINANT
5G protein beta subunit (Gb1)COMPLEX1RECOMBINANT
6G protein gamma subunit 2 (Gg2)COMPLEX1RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Homo sapiens (human)9606
65Homo sapiens (human)9606
76Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Trichoplusia ni (cabbage looper)7111
32Trichoplusia ni (cabbage looper)7111
43Trichoplusia ni (cabbage looper)7111
54Trichoplusia ni (cabbage looper)7111
65Trichoplusia ni (cabbage looper)7111
76Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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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 defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingAverage exposure time: 2 sec. / Electron dose: 69.14 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2Leginonimage acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
9RELION3.1initial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3993932
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53984 / Symmetry type: POINT

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