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- PDB-9ax5: Cryo-EM structure of Phospholipase C epsilon PH-C terminus in com... -

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Basic information

Entry
Database: PDB / ID: 9ax5
TitleCryo-EM structure of Phospholipase C epsilon PH-C terminus in complex with RhoA-GTP
Components
  • 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1
  • Transforming protein RhoA
KeywordsMEMBRANE PROTEIN / GPCR signaling / complex / phospholipase / PIP2 hydrolysis / G protein
Function / homology
Function and homology information


diacylglycerol biosynthetic process / Synthesis of IP3 and IP4 in the cytosol / phosphoinositide phospholipase C / alpha-beta T cell lineage commitment / aortic valve formation / mitotic cleavage furrow formation / positive regulation of lipase activity / bone trabecula morphogenesis / endothelial tube lumen extension / skeletal muscle satellite cell migration ...diacylglycerol biosynthetic process / Synthesis of IP3 and IP4 in the cytosol / phosphoinositide phospholipase C / alpha-beta T cell lineage commitment / aortic valve formation / mitotic cleavage furrow formation / positive regulation of lipase activity / bone trabecula morphogenesis / endothelial tube lumen extension / skeletal muscle satellite cell migration / positive regulation of vascular associated smooth muscle contraction / angiotensin-mediated vasoconstriction involved in regulation of systemic arterial blood pressure / SLIT2:ROBO1 increases RHOA activity / RHO GTPases Activate Rhotekin and Rhophilins / Roundabout signaling pathway / negative regulation of intracellular steroid hormone receptor signaling pathway / phosphatidylinositol metabolic process / Axonal growth inhibition (RHOA activation) / Axonal growth stimulation / cleavage furrow formation / regulation of neural precursor cell proliferation / regulation of modification of postsynaptic actin cytoskeleton / regulation of osteoblast proliferation / forebrain radial glial cell differentiation / phospholipase C activity / phosphatidylinositol-4,5-bisphosphate phospholipase C activity / glomerulus development / apical junction assembly / cell junction assembly / negative regulation of cell migration involved in sprouting angiogenesis / cellular response to chemokine / establishment of epithelial cell apical/basal polarity / beta selection / regulation of systemic arterial blood pressure by endothelin / negative regulation of cell size / negative regulation of oxidative phosphorylation / negative regulation of motor neuron apoptotic process / RHO GTPases Activate ROCKs / regulation of modification of postsynaptic structure / RHO GTPases activate CIT / Sema4D induced cell migration and growth-cone collapse / PCP/CE pathway / RHO GTPases activate KTN1 / positive regulation of podosome assembly / apolipoprotein A-I-mediated signaling pathway / positive regulation of alpha-beta T cell differentiation / Sema4D mediated inhibition of cell attachment and migration / positive regulation of leukocyte adhesion to vascular endothelial cell / wound healing, spreading of cells / motor neuron apoptotic process / PI3K/AKT activation / odontogenesis / Wnt signaling pathway, planar cell polarity pathway / ossification involved in bone maturation / regulation of focal adhesion assembly / negative chemotaxis / apical junction complex / EPHA-mediated growth cone collapse / phosphatidylinositol-mediated signaling / myosin binding / regulation of neuron projection development / stress fiber assembly / cellular response to cytokine stimulus / RHOC GTPase cycle / positive regulation of cytokinesis / cerebral cortex cell migration / ERBB2 Regulates Cell Motility / cleavage furrow / semaphorin-plexin signaling pathway / androgen receptor signaling pathway / ficolin-1-rich granule membrane / RHOA GTPase cycle / mitotic spindle assembly / negative regulation of cell-substrate adhesion / Rho protein signal transduction / positive regulation of protein serine/threonine kinase activity / endothelial cell migration / positive regulation of T cell migration / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / skeletal muscle tissue development / lipid catabolic process / positive regulation of lamellipodium assembly / RHO GTPases activate PKNs / GPVI-mediated activation cascade / negative regulation of reactive oxygen species biosynthetic process / positive regulation of stress fiber assembly / cytoplasmic microtubule organization / release of sequestered calcium ion into cytosol / EPHB-mediated forward signaling / substantia nigra development / positive regulation of neuron differentiation / substrate adhesion-dependent cell spreading / regulation of microtubule cytoskeleton organization / regulation of cell migration / secretory granule membrane / guanyl-nucleotide exchange factor activity / small monomeric GTPase / cell-matrix adhesion / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / kidney development
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 / Small GTPase Rho / small GTPase Rho family profile. / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain / C2 domain profile. / C2 domain superfamily / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / EF-hand domain pair / Small GTP-binding protein domain / Ubiquitin-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / Transforming protein RhoA / 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsOhri, V. / Lyon, A.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)1R01HL141076-01 United States
CitationJournal: To Be Published
Title: Cryo-EM structure of Phospholipase C epsilon PH-C terminus in complex with RhoA-GTP
Authors: Ohri, V. / Lyon, A.M.
History
DepositionMar 5, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Primary map / Data content type: Primary map / Provider: 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 epsilon-1
C: Transforming protein RhoA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)190,3405
Polymers189,7522
Non-polymers5883
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 165182.938 Da / Num. of mol.: 1 / Fragment: PH-C terminal residues 837-2281
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Plce1, Plce / Plasmid: pFastBacHTA / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q99P84, phosphoinositide phospholipase C
#2: Protein Transforming protein RhoA / Rho cDNA clone 12 / h12


Mass: 24569.055 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RHOA, ARH12, ARHA, RHO12 / Plasmid: pFastBacHTA / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P61586, small monomeric GTPase
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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
1PLCe PH-C in complex with RhoA-GTPCOMPLEX#1-#20MULTIPLE SOURCES
2PH-C fragment (837-2281) of PLC epsilonCOMPLEX#11RECOMBINANT
3RhoA-GTPCOMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.189 MDaNO
210.165 MDaNO
310.024 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Rattus norvegicus (Norway rat)10116
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
12Spodoptera frugiperda (fall armyworm)7108pFastBacHTA
23Trichoplusia ni (cabbage looper)7111pFastBacHTA
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2150 mMsodium chlorideNaCl1
32 mMdithiothreitolDTT1
40.1 mMethylenediaminetetraacetic acidEDTA1
50.1 mMegtazic acidEGTA1
61 mMmagnesium dichlorideMgCl21
70.04 mMguanosine triphosphateGTP1
80.5 mMcalcium chlorideCaCl21
92.5 mM3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonateCHAPS1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.2 sec. / Electron dose: 57.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6345

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1329298
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 209463 / Symmetry type: POINT

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