+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-25153 | |||||||||
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Title | Human Trio residues 1284-1959 in complex with Rac1 | |||||||||
Map data | TrioN-Rac1 complex | |||||||||
Sample |
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Function / homology | Function and homology information cell surface receptor protein tyrosine phosphatase signaling pathway / regulation of respiratory burst / negative regulation of interleukin-23 production / regulation of neutrophil migration / localization within membrane / Activated NTRK2 signals through CDK5 / NADPH oxidase complex / negative regulation of receptor-mediated endocytosis / regulation of hydrogen peroxide metabolic process / ruffle assembly ...cell surface receptor protein tyrosine phosphatase signaling pathway / regulation of respiratory burst / negative regulation of interleukin-23 production / regulation of neutrophil migration / localization within membrane / Activated NTRK2 signals through CDK5 / NADPH oxidase complex / negative regulation of receptor-mediated endocytosis / regulation of hydrogen peroxide metabolic process / ruffle assembly / NTRK2 activates RAC1 / engulfment of apoptotic cell / Inactivation of CDC42 and RAC1 / WNT5:FZD7-mediated leishmania damping / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / cortical cytoskeleton organization / respiratory burst / hepatocyte growth factor receptor signaling pathway / ruffle organization / cell projection assembly / negative regulation of fibroblast migration / thioesterase binding / regulation of stress fiber assembly / RHO GTPases activate CIT / Nef and signal transduction / sphingosine-1-phosphate receptor signaling pathway / regulation of nitric oxide biosynthetic process / PCP/CE pathway / Azathioprine ADME / motor neuron axon guidance / RHO GTPases activate KTN1 / positive regulation of neutrophil chemotaxis / regulation of lamellipodium assembly / Activation of RAC1 / DCC mediated attractive signaling / positive regulation of cell-substrate adhesion / MET activates RAP1 and RAC1 / regulation of small GTPase mediated signal transduction / Wnt signaling pathway, planar cell polarity pathway / Sema4D mediated inhibition of cell attachment and migration / CD28 dependent Vav1 pathway / Ephrin signaling / lamellipodium assembly / negative regulation of fat cell differentiation / positive regulation of Rho protein signal transduction / establishment or maintenance of cell polarity / regulation of cell size / DSCAM interactions / Activation of RAC1 downstream of NMDARs / extrinsic component of membrane / presynaptic active zone / Rho GDP-dissociation inhibitor binding / small GTPase-mediated signal transduction / postsynaptic modulation of chemical synaptic transmission / NRAGE signals death through JNK / Rac protein signal transduction / RHOJ GTPase cycle / RHO GTPases activate PAKs / positive regulation of focal adhesion assembly / semaphorin-plexin signaling pathway / CDC42 GTPase cycle / Sema3A PAK dependent Axon repulsion / ficolin-1-rich granule membrane / RHOG GTPase cycle / EPH-ephrin mediated repulsion of cells / RHOA GTPase cycle / RHO GTPases Activate NADPH Oxidases / anatomical structure morphogenesis / RAC3 GTPase cycle / RAC2 GTPase cycle / RHO GTPases Activate WASPs and WAVEs / RHO GTPases activate IQGAPs / localization / positive regulation of lamellipodium assembly / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / positive regulation of substrate adhesion-dependent cell spreading / regulation of cell migration / RHO GTPases activate PKNs / positive regulation of stress fiber assembly / GPVI-mediated activation cascade / RAC1 GTPase cycle / EPHB-mediated forward signaling / actin filament polymerization / positive regulation of microtubule polymerization / cell-matrix adhesion / cell chemotaxis / guanyl-nucleotide exchange factor activity / substrate adhesion-dependent cell spreading / small monomeric GTPase / neuron projection morphogenesis / G protein activity / positive regulation of endothelial cell migration / secretory granule membrane / VEGFR2 mediated vascular permeability / Signal transduction by L1 / cell projection / cell motility / actin filament organization / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.86 Å | |||||||||
Authors | Chen C-L / Ravala SK / Bandekar SJ / Cash J / Tesmer JJG | |||||||||
Funding support | United States, 2 items
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Citation | Journal: J Biol Chem / Year: 2022 Title: Structural/functional studies of Trio provide insights into its configuration and show that conserved linker elements enhance its activity for Rac1. Authors: Sumit J Bandekar / Chun-Liang Chen / Sandeep K Ravala / Jennifer N Cash / Larisa V Avramova / Mariya V Zhalnina / J Silvio Gutkind / Sheng Li / John J G Tesmer / Abstract: Trio is a large and highly conserved metazoan signaling scaffold that contains two Dbl family guanine nucleotide exchange factor (GEF) modules, TrioN and TrioC, selective for Rac and RhoA GTPases, ...Trio is a large and highly conserved metazoan signaling scaffold that contains two Dbl family guanine nucleotide exchange factor (GEF) modules, TrioN and TrioC, selective for Rac and RhoA GTPases, respectively. The GEF activities of TrioN and TrioC are implicated in several cancers, especially uveal melanoma. However, little is known about how these modules operate in the context of larger fragments of Trio. Here we show via negative stain electron microscopy that the N-terminal region of Trio is extended and could thus serve as a rigid spacer between the N-terminal putative lipid-binding domain and TrioN, whereas the C-terminal half of Trio seems globular. We found that regions C-terminal to TrioN enhance its Rac1 GEF activity and thus could play a regulatory role. We went on to characterize a minimal, well-behaved Trio fragment with enhanced activity, Trio, in complex with Rac1 using cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry and found that the region conferring enhanced activity is disordered. Deletion of two different strongly conserved motifs in this region eliminated this enhancement, suggesting that they form transient intramolecular interactions that promote GEF activity. Because Dbl family RhoGEF modules have been challenging to directly target with small molecules, characterization of accessory Trio domains such as these may provide alternate routes for the development of therapeutics that inhibit Trio activity in human cancer. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_25153.map.gz | 28.7 MB | EMDB map data format | |
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Header (meta data) | emd-25153-v30.xml emd-25153.xml | 18 KB 18 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_25153_fsc.xml | 6.9 KB | Display | FSC data file |
Images | emd_25153.png | 56.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-25153 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-25153 | HTTPS FTP |
-Related structure data
Related structure data | 7sj4MC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_25153.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | TrioN-Rac1 complex | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.08 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Sample components
-Entire : Human Trio residues 1284-1959 in complex with Rac1
Entire | Name: Human Trio residues 1284-1959 in complex with Rac1 |
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Components |
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-Supramolecule #1: Human Trio residues 1284-1959 in complex with Rac1
Supramolecule | Name: Human Trio residues 1284-1959 in complex with Rac1 / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: Rosetta (DE3) pLysS |
-Macromolecule #1: Triple functional domain protein
Macromolecule | Name: Triple functional domain protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 75.592969 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: SNAEEKRKSA RRKEFIMAEL IQTEKAYVRD LRECMDTYLW EMTSGVEEIP PGIVNKELII FGNMQEIYEF HNNIFLKELE KYEQLPEDV GHCFVTWADK FQMYVTYCKN KPDSTQLILE HAGSYFDEIQ QRHGLANSIS SYLIKPVQRI TKYQLLLKEL L TCCEEGKG ...String: SNAEEKRKSA RRKEFIMAEL IQTEKAYVRD LRECMDTYLW EMTSGVEEIP PGIVNKELII FGNMQEIYEF HNNIFLKELE KYEQLPEDV GHCFVTWADK FQMYVTYCKN KPDSTQLILE HAGSYFDEIQ QRHGLANSIS SYLIKPVQRI TKYQLLLKEL L TCCEEGKG EIKDGLEVML SVPKRANDAM HLSMLEGFDE NIESQGELIL QESFQVWDPK TLIRKGRERH LFLFEMSLVF SK EVKDSSG RSKYLYKSKL FTSELGVTEH VEGDPCKFAL WVGRTPTSDN KIVLKASSIE NKQDWIKHIR EVIQERTIHL KGA LKEPIH IPKTAPATRQ KGRRDGEDLD SQGDGSSQPD TISIASRTSQ NTLDSDKLSG GCELTVVIHD FTACNSNELT IRRG QTVEV LERPHDKPDW CLVRTTDRSP AAEGLVPCGS LCIAHSRSSM EMEGIFNHKD SLSVSSNDAS PPASVASLQP HMIGA QSSP GPKRPGNTLR KWLTSPVRRL SSGKADGHVK KLAHKHKKSR EVRKSADAGS QKDSDDSAAT PQDETVEERG RNEGLS SGT LSKSSSSGMQ SCGEEEGEEG ADAVPLPPPM AIQQHSLLQP DSQDDKASSR LLVRPTSSET PSAAELVSAI EELVKSK MA LEDRPSSLLV DQGDSSSPSF NPSDNSLLSS SSPIDEM |
-Macromolecule #2: Ras-related C3 botulinum toxin substrate 1
Macromolecule | Name: Ras-related C3 botulinum toxin substrate 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 21.811453 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: GEFMQAIKCV VVGDGAVGKT CLLISYTTNA FPGEYIPTVF DNYSANVMVD GKPVNLGLWD TAGQEDYDRL RPLSYPQTDV FLICFSLVS PASFENVRAK WYPEVRHHCP NTPIILVGTK LDLRDDKDTI EKLKEKKLTP ITYPQGLAMA KEIGAVKYLE C SALTQRGL ...String: GEFMQAIKCV VVGDGAVGKT CLLISYTTNA FPGEYIPTVF DNYSANVMVD GKPVNLGLWD TAGQEDYDRL RPLSYPQTDV FLICFSLVS PASFENVRAK WYPEVRHHCP NTPIILVGTK LDLRDDKDTI EKLKEKKLTP ITYPQGLAMA KEIGAVKYLE C SALTQRGL KTVFDEAIRA VLCPPPVKKR KRKCLLL |
-Macromolecule #3: water
Macromolecule | Name: water / type: ligand / ID: 3 / Number of copies: 24 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.25 mg/mL | ||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force 10. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Alignment procedure | Coma free - Residual tilt: 0.01 mrad |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number real images: 2817 / Average exposure time: 3.12 sec. / Average electron dose: 55.0 e/Å2 Details: 3514 movies were initially collected. After motion correction and CTF estimation, 697 micrographs were rejected due to poor CTF fitting. |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
-Atomic model buiding 1
Initial model |
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Details | The initial model was generated from the pdb structure (2NZ8) using the SWISS-MODEL server and rigid-body fitted using Chimera. Several runs of structure refinement were done using the coot and phenix real-space refinement. | ||||||
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient | ||||||
Output model | PDB-7sj4: |