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- PDB-8tua: Full-length P-Rex1 in complex with inositol 1,3,4,5-tetrakisphosp... -

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Entry
Database: PDB / ID: 8tua
TitleFull-length P-Rex1 in complex with inositol 1,3,4,5-tetrakisphosphate (IP4)
ComponentsPhosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein
KeywordsSIGNALING PROTEIN / Rho guanine-nucleotide exchange factor / Dbl homology domain / pleckstrin homology domain / Phosphatidylinositol 3 / 4 / 5-trisphosphate binding
Function / homology
Function and homology information


regulation of signaling / regulation of dendrite development / neutrophil activation / regulation of actin filament polymerization / regulation of small GTPase mediated signal transduction / RHOB GTPase cycle / NRAGE signals death through JNK / superoxide metabolic process / RHOJ GTPase cycle / RHOC GTPase cycle ...regulation of signaling / regulation of dendrite development / neutrophil activation / regulation of actin filament polymerization / regulation of small GTPase mediated signal transduction / 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 / guanyl-nucleotide exchange factor activity / neutrophil chemotaxis / dendritic shaft / phospholipid binding / G alpha (12/13) signalling events / growth cone / intracellular signal transduction / positive regulation of cell migration / G protein-coupled receptor signaling pathway / perinuclear region of cytoplasm / enzyme binding / plasma membrane / cytosol
Similarity search - Function
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 ...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. / 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 / PH-like domain superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
INOSITOL-(1,3,4,5)-TETRAKISPHOSPHATE / Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsCash, J.N. / Tesmer, J.J.G.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA254402 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA221289 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL071818 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P30CA023168 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM146664 United States
Citation
Journal: Elife / Year: 2024
Title: Full-length P-Rex1 in complex with inositol 1,3,4,5-tetrakisphosphate (IP4)
Authors: Ravala, S.K. / Adame-Garcia, S.R. / Li, S. / Chen, C. / Cianfrocco, M.A. / Gutkind, J.S. / Cash, J.N. / Tesmer, J.J.G.
#1: Journal: Acta Crystallogr D Struct Biol / Year: 2019
Title: Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.
Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty ...Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty / Robert D Oeffner / Billy K Poon / Michael G Prisant / Randy J Read / Jane S Richardson / David C Richardson / Massimo D Sammito / Oleg V Sobolev / Duncan H Stockwell / Thomas C Terwilliger / Alexandre G Urzhumtsev / Lizbeth L Videau / Christopher J Williams / Paul D Adams /
Abstract: Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological ...Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.
#2: Journal: Nat Methods / Year: 2019
Title: Real-time cryo-electron microscopy data preprocessing with Warp.
Authors: Dimitry Tegunov / Patrick Cramer /
Abstract: The acquisition of cryo-electron microscopy (cryo-EM) data from biological specimens must be tightly coupled to data preprocessing to ensure the best data quality and microscope usage. Here we ...The acquisition of cryo-electron microscopy (cryo-EM) data from biological specimens must be tightly coupled to data preprocessing to ensure the best data quality and microscope usage. Here we describe Warp, a software that automates all preprocessing steps of cryo-EM data acquisition and enables real-time evaluation. Warp corrects micrographs for global and local motion, estimates the local defocus and monitors key parameters for each recorded micrograph or tomographic tilt series in real time. The software further includes deep-learning-based models for accurate particle picking and image denoising. The output from Warp can be fed into established programs for particle classification and 3D-map refinement. Our benchmarks show improvement in the nominal resolution, which went from 3.9 Å to 3.2 Å, of a published cryo-EM data set for influenza virus hemagglutinin. Warp is easy to install from http://github.com/cramerlab/warp and computationally inexpensive, and has an intuitive, streamlined user interface.
#3: Journal: Nat Methods / Year: 2020
Title: Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction.
Authors: Ali Punjani / Haowei Zhang / David J Fleet /
Abstract: Cryogenic electron microscopy (cryo-EM) is widely used to study biological macromolecules that comprise regions with disorder, flexibility or partial occupancy. For example, membrane proteins are ...Cryogenic electron microscopy (cryo-EM) is widely used to study biological macromolecules that comprise regions with disorder, flexibility or partial occupancy. For example, membrane proteins are often kept in solution with detergent micelles and lipid nanodiscs that are locally disordered. Such spatial variability negatively impacts computational three-dimensional (3D) reconstruction with existing iterative refinement algorithms that assume rigidity. We introduce non-uniform refinement, an algorithm based on cross-validation optimization, which automatically regularizes 3D density maps during refinement to account for spatial variability. Unlike common shift-invariant regularizers, non-uniform refinement systematically removes noise from disordered regions, while retaining signal useful for aligning particle images, yielding dramatically improved resolution and 3D map quality in many cases. We obtain high-resolution reconstructions for multiple membrane proteins as small as 100 kDa, demonstrating increased effectiveness of cryo-EM for this class of targets critical in structural biology and drug discovery. Non-uniform refinement is implemented in the cryoSPARC software package.
History
DepositionAug 15, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 10, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)183,9602
Polymers183,4591
Non-polymers5001
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein


Mass: 183459.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Inositol 1,3,4,5-tetrakisphosphate / Source: (gene. exp.) Homo sapiens (human) / Gene: PREX1 / Cell line (production host): 293 Freestyle / Production host: Homo sapiens (human) / References: UniProt: Q8TCU6
#2: Chemical ChemComp-4IP / INOSITOL-(1,3,4,5)-TETRAKISPHOSPHATE


Mass: 500.075 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H16O18P4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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

ComponentName: Complex of full-length Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) bound to inositol 1,3,4,5-tetrakisphosphate (IP4)
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.183 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293 Freestyle
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
2100 mMsodium chlorideNaClSodium chloride1
32 mMDTT1
SpecimenConc.: 0.56 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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 200 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 57.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21_5207 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2426612
Details: 806,067 particles untilted and 1,620,545 particles tilted
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 187734 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Chain-ID: A / Pdb chain-ID: A / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeInitial refinement model-ID
16PCV

6pcv

6PCV1
26VSK

6vsk

6VSK2
35D3X

5d3x

5D3X3
45FI1

5fi1

5FI14
57RX9

7rx9

7RX95
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 103.39 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004210876
ELECTRON MICROSCOPYf_angle_d0.558414670
ELECTRON MICROSCOPYf_chiral_restr0.03651640
ELECTRON MICROSCOPYf_plane_restr0.00391883
ELECTRON MICROSCOPYf_dihedral_angle_d12.95984153

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