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- PDB-6pcv: Single Particle Reconstruction of Phosphatidylinositol (3,4,5) tr... -

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Basic information

Entry
Database: PDB / ID: 6pcv
TitleSingle Particle Reconstruction of Phosphatidylinositol (3,4,5) trisphosphate-dependent Rac exchanger 1 bound to G protein beta gamma subunits
Components
  • 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
  • Phosphatidylinositol (3,4,5) trisphosphate-dependent Rac exchanger 1
KeywordsSIGNALING PROTEIN / RhoGEF / G protein / Complex / Phosphatase fold
Function / homology
Function and homology information


regulation of signaling / Olfactory Signaling Pathway / regulation of dendrite development / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / regulation of actin filament polymerization / Activation of the phototransduction cascade / neutrophil activation / Activation of G protein gated Potassium channels / G-protein activation ...regulation of signaling / Olfactory Signaling Pathway / regulation of dendrite development / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / regulation of actin filament polymerization / Activation of the phototransduction cascade / neutrophil activation / Activation of G protein gated Potassium channels / G-protein activation / G beta:gamma signalling through PI3Kgamma / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through PLC beta / ADP signalling through P2Y purinoceptor 1 / Thromboxane signalling through TP receptor / Presynaptic function of Kainate receptors / G beta:gamma signalling through CDC42 / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / G alpha (12/13) signalling events / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / regulation of small GTPase mediated signal transduction / Thrombin signalling through proteinase activated receptors (PARs) / Ca2+ pathway / G alpha (z) signalling events / Extra-nuclear estrogen signaling / G alpha (s) signalling events / G alpha (q) signalling events / G alpha (i) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / RHOB GTPase cycle / NRAGE signals death through JNK / superoxide metabolic process / RHOJ GTPase cycle / RHOC GTPase cycle / RHOQ GTPase cycle / CDC42 GTPase cycle / RHOG GTPase cycle / T cell differentiation / RHOA GTPase cycle / RAC2 GTPase cycle / RAC3 GTPase cycle / positive regulation of substrate adhesion-dependent cell spreading / RAC1 GTPase cycle / actin filament polymerization / GTPase activator activity / neutrophil chemotaxis / guanyl-nucleotide exchange factor activity / dendritic shaft / phospholipid binding / photoreceptor disc membrane / cellular response to catecholamine stimulus / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / signaling receptor complex adaptor activity / growth cone / intracellular signal transduction / positive regulation of cell migration / G protein-coupled receptor signaling pathway / GTPase activity / perinuclear region of cytoplasm / enzyme binding / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / Domain found in Dishevelled, Egl-10, and Pleckstrin (DEP) / DEP domain profile. / Domain found in Dishevelled, Egl-10, and Pleckstrin / DEP domain / Guanine-nucleotide dissociation stimulator, CDC24, conserved site / Dbl homology (DH) domain signature. / Dbl homology (DH) domain superfamily / RhoGEF domain ...Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / Domain found in Dishevelled, Egl-10, and Pleckstrin (DEP) / DEP domain profile. / Domain found in Dishevelled, Egl-10, and Pleckstrin / DEP domain / Guanine-nucleotide dissociation stimulator, CDC24, conserved site / Dbl homology (DH) domain signature. / Dbl homology (DH) domain superfamily / RhoGEF domain / Guanine nucleotide exchange factor for Rho/Rac/Cdc42-like GTPases / Dbl homology (DH) domain / Dbl homology (DH) domain profile. / YVTN repeat-like/Quinoprotein amine dehydrogenase / 7 Propeller / Methylamine Dehydrogenase; Chain H / PH domain profile. / Pleckstrin homology domain. / Pleckstrin homology domain / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ 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 / Few Secondary Structures / Irregular / PH-like domain superfamily / G-protein beta WD-40 repeat / Winged helix DNA-binding domain superfamily / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Mainly Beta
Similarity search - Domain/homology
PREX1 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsCash, J.N. / Cianfrocco, M.A. / Tesmer, J.J.G.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA221289 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL122416 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL071818 United States
CitationJournal: Sci Adv / Year: 2019
Title: Cryo-electron microscopy structure and analysis of the P-Rex1-Gβγ signaling scaffold.
Authors: Jennifer N Cash / Sarah Urata / Sheng Li / Sandeep K Ravala / Larisa V Avramova / Michael D Shost / J Silvio Gutkind / John J G Tesmer / Michael A Cianfrocco /
Abstract: PIP-dependent Rac exchanger 1 (P-Rex1) is activated downstream of G protein-coupled receptors to promote neutrophil migration and metastasis. The structure of more than half of the enzyme and its ...PIP-dependent Rac exchanger 1 (P-Rex1) is activated downstream of G protein-coupled receptors to promote neutrophil migration and metastasis. The structure of more than half of the enzyme and its regulatory G protein binding site are unknown. Our 3.2 Å cryo-EM structure of the P-Rex1-Gβγ complex reveals that the carboxyl-terminal half of P-Rex1 adopts a complex fold most similar to those of phosphoinositide phosphatases. Although catalytically inert, the domain coalesces with a DEP domain and two PDZ domains to form an extensive docking site for Gβγ. Hydrogen-deuterium exchange mass spectrometry suggests that Gβγ binding induces allosteric changes in P-Rex1, but functional assays indicate that membrane localization is also required for full activation. Thus, a multidomain assembly is key to the regulation of P-Rex1 by Gβγ and the formation of a membrane-localized scaffold optimized for recruitment of other signaling proteins such as PKA and PTEN.
History
DepositionJun 18, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 23, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Phosphatidylinositol (3,4,5) trisphosphate-dependent Rac exchanger 1
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


Theoretical massNumber of molelcules
Total (without water)231,4843
Polymers231,4843
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Phosphatidylinositol (3,4,5) trisphosphate-dependent Rac exchanger 1 / PREX1 / RAC EXCHANGER 1


Mass: 184840.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PREX1 / Cell line (production host): 293F / Production host: Homo sapiens (human) / References: UniProt: A0A2X0SFH1, UniProt: Q8TCU6*PLUS
#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.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Gene: GNB1 / Cell line (production host): BTI-Tn-5B1-4 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P62871
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 9226.547 Da / Num. of mol.: 1 / Mutation: C68S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Gene: GNG2 / Cell line (production host): BTI-Tn-5B1-4 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63212

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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
1P-Rex1 bound to G protein beta gamma subunitsCOMPLEXall0MULTIPLE SOURCES
2Phosphatidylinositol (3,4,5) trisphosphate-dependent Rac exchanger 1COMPLEX#11MULTIPLE SOURCES
3G protein beta-1 subunitCOMPLEX#21MULTIPLE SOURCES
4G protein gamma-2 subunitCOMPLEX#31MULTIPLE SOURCES
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.231 MDaYES
210.185 MDaYES
310.037 MDaYES
410.009 MDaYES
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, 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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 29000 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 47 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 6746
Image scansWidth: 3838 / Height: 3710

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.14_3260refinement
PHENIX1.14_3260refinement
EM software
IDNameVersionCategory
2Leginon3.2image acquisition
4Warp1.0.6CTF correction
7PHENIX1.14-3260model fitting
9PHENIX1.14-3260model refinement
10cryoSPARCv0.65initial Euler assignment
11cryoSPARCv0.65final Euler assignment
12cryoSPARCv0.65classification
13cryoSPARCv0.653D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 905464
Details: 600,588 particles (untilted) and 304,876 particles (30 degree tilted)
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 205599 / Symmetry type: POINT
Atomic model buildingB value: 83 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-IDPdb chain residue range
13V5W

3v5w

B3V5W1
23V5W

3v5w

G3V5W1
33QIK

3qik

A3QIK2622-706
RefinementStereochemistry target values: CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00589202
ELECTRON MICROSCOPYf_angle_d0.966912445
ELECTRON MICROSCOPYf_chiral_restr0.05921392
ELECTRON MICROSCOPYf_plane_restr0.00671621
ELECTRON MICROSCOPYf_dihedral_angle_d8.99075586

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