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- PDB-9g5i: Cryo-EM structure of a 2:1 ALK:ALKAL2 complex obtained after re-p... -

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Basic information

Entry
Database: PDB / ID: 9g5i
TitleCryo-EM structure of a 2:1 ALK:ALKAL2 complex obtained after re-processing of EMPIAR-10930 data
Components
  • ALK and LTK ligand 2
  • ALK tyrosine kinase receptor
KeywordsCYTOKINE / Complex / receptor tyrosine kinase
Function / homology
Function and homology information


positive regulation of ERK5 cascade / ASP-3026-resistant ALK mutants / NVP-TAE684-resistant ALK mutants / alectinib-resistant ALK mutants / brigatinib-resistant ALK mutants / ceritinib-resistant ALK mutants / crizotinib-resistant ALK mutants / lorlatinib-resistant ALK mutants / MDK and PTN in ALK signaling / receptor signaling protein tyrosine kinase activator activity ...positive regulation of ERK5 cascade / ASP-3026-resistant ALK mutants / NVP-TAE684-resistant ALK mutants / alectinib-resistant ALK mutants / brigatinib-resistant ALK mutants / ceritinib-resistant ALK mutants / crizotinib-resistant ALK mutants / lorlatinib-resistant ALK mutants / MDK and PTN in ALK signaling / receptor signaling protein tyrosine kinase activator activity / regulation of dopamine receptor signaling pathway / response to environmental enrichment / ALK mutants bind TKIs / transmembrane receptor protein tyrosine kinase activator activity / swimming behavior / phosphorylation / Signaling by LTK / positive regulation of dendrite development / regulation of neuron differentiation / peptidyl-tyrosine autophosphorylation / Signaling by ALK / response to stress / adult behavior / energy homeostasis / neuron development / negative regulation of lipid catabolic process / transmembrane receptor protein tyrosine kinase activity / cell surface receptor protein tyrosine kinase signaling pathway / cytokine activity / hippocampus development / positive regulation of NF-kappaB transcription factor activity / receptor protein-tyrosine kinase / positive regulation of neuron projection development / receptor tyrosine kinase binding / Signaling by ALK fusions and activated point mutants / heparin binding / regulation of cell population proliferation / protein autophosphorylation / protein tyrosine kinase activity / regulation of apoptotic process / positive regulation of ERK1 and ERK2 cascade / receptor complex / signal transduction / protein-containing complex / extracellular space / extracellular exosome / extracellular region / ATP binding / identical protein binding / plasma membrane
Similarity search - Function
ALK and LTK ligand 1/2 / ALK and LTK ligand 1/2 / : / ALK/LTK, Glycine-rich domain / MAM domain, meprin/A5/mu / MAM domain / MAM domain profile. / Tyrosine-protein kinase, receptor class II, conserved site / Receptor tyrosine kinase class II signature. / Low-density lipoprotein receptor domain class A ...ALK and LTK ligand 1/2 / ALK and LTK ligand 1/2 / : / ALK/LTK, Glycine-rich domain / MAM domain, meprin/A5/mu / MAM domain / MAM domain profile. / Tyrosine-protein kinase, receptor class II, conserved site / Receptor tyrosine kinase class II signature. / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A repeat / LDL receptor-like superfamily / : / Tyrosine-protein kinase, catalytic domain / Tyrosine kinase, catalytic domain / Tyrosine protein kinases specific active-site signature. / Tyrosine-protein kinase, active site / Concanavalin A-like lectin/glucanase domain superfamily / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein tyrosine and serine/threonine kinase / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
ALK and LTK ligand 2 / ALK tyrosine kinase receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsFelix, J. / De Munck, S. / Bazan, J.F. / Savvides, S.N.
Funding support Belgium, 2items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)G0B4918N Belgium
Other governmentFlanders Institute for Biotechnology (VIB) grant number C0101 Belgium
CitationJournal: PLoS Biol / Year: 2025
Title: Reanalysis of cryo-EM data reveals ALK-cytokine assemblies with both 2:1 and 2:2 stoichiometries.
Authors: Jan Felix / Steven De Munck / J Fernando Bazan / Savvas N Savvides /
Abstract: Activation of Anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK) by their cognate cytokines ALKAL2 and ALKAL1 plays important roles in development, metabolism, and cancer. Recent ...Activation of Anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK) by their cognate cytokines ALKAL2 and ALKAL1 plays important roles in development, metabolism, and cancer. Recent structural studies revealed ALK/LTK-cytokine assemblies with distinct stoichiometries. Structures of ALK-ALKAL2 and LTK-ALKAL1 complexes with 2:1 stoichiometry determined by X-ray crystallography contrasted the 2:2 ALK-ALKAL2 complexes determined by cryo-EM and X-ray crystallography. Here, we show based on reanalysis of the cryo-EM data deposited in EMPIAR-10930 that over half of the ALK-ALKAL2 particles in the dataset are classified into 2D and 3D classes obeying a 2:1 stoichiometry besides the originally reported structure displaying 2:2 stoichiometry. Unlike particles representing the 2:2 ALK-ALKAL2 complex, particles for the 2:1 ALK-ALKAL2 complex suffer severely from preferred orientations that resulted in cryo-EM maps displaying strong anisotropy. Here, we show that extensive particle orientation rebalancing in cryoSPARC followed by 3D refinement with Blush regularization in RELION constitutes an effective strategy for avoiding map artifacts relating to preferred particle orientations and report a 3D reconstruction of the 2:1 ALK-ALKAL2 complex to 3.2 Å resolution from EMPIAR-10930. This new cryo-EM structure together with the crystal structures of ALK-ALKAL2 and LTK-ALKAL1 complexes with 2:1 stoichiometry reconciles a common receptor dimerization mode for ALK and LTK and provides direct evidence for the presence of an ALK-ALKAL2 complex with 2:1 stoichiometry next to the reported 2:2 stoichiometric assembly in the EMPIAR-10930 dataset. Finally, our analysis emphasizes the importance of public deposition of raw cryo-EM data to allow reanalysis and interpretation.
History
DepositionJul 17, 2024Deposition site: PDBE / Processing site: PDBE
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: Additional map / Part number: 1 / Data content type: Additional map / 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
Revision 1.1Apr 23, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ALK tyrosine kinase receptor
B: ALK tyrosine kinase receptor
C: ALK and LTK ligand 2


Theoretical massNumber of molelcules
Total (without water)93,3953
Polymers93,3953
Non-polymers00
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 ALK tyrosine kinase receptor / Anaplastic lymphoma kinase


Mass: 39377.559 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ALK / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q9UM73, receptor protein-tyrosine kinase
#2: Protein ALK and LTK ligand 2 / Augmentor alpha / AUG-alpha


Mass: 14640.013 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ALKAL2, FAM150B, UNQ542/PRO1097 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q6UX46
Has protein modificationY

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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: Cryo-EM structure of a 2:1 ALK:ALKAL2 complex obtained after re-processing of EMPIAR-10930 data
Type: COMPLEX
Details: Sample preparation and data collection was performed by Reshetnyak et al. (Nature, 2021, https://doi.org/10.1038/s41586-021-04140-8). Motion corrected micrographs and an uncleaned particle ...Details: Sample preparation and data collection was performed by Reshetnyak et al. (Nature, 2021, https://doi.org/10.1038/s41586-021-04140-8). Motion corrected micrographs and an uncleaned particle stack containing 18,053,750 particles were downloaded from EMPIAR entry 10930. All subsequent data processing, refinement en model building was performed by Jan Felix with help from Steven De Munck and Savvas Savvides.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.086 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
115 mMHEPES1
2150 mMNaCl1
33 %PEG40001
SpecimenConc.: 1 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 R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283.15 K
Details: QUANTIFOIL R1.2/1/3 gold 300 mesh grids without glow-discharge, blotting time 3 sec., blotting force -5.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: SerialEM coma-free alignment
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 98 K / Temperature (min): 96 K
Image recordingAverage exposure time: 4.2 sec. / Electron dose: 81 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13618
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3.1particle selection
4cryoSPARC3.3.1CTF correction
7UCSF Chimeramodel fitting
9cryoSPARC4.5initial Euler assignment
10cryoSPARC4.5final Euler assignment
11cryoSPARC4.5classification
12RELION53D reconstruction
13PHENIX1.19.2-4158model refinement
14REFMACmodel refinementRefmac-servalcat from CCP-EM v1.6
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 18053705
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142986
Details: Final 3D refinement was performed in Relion v5 with enabled Blush Regularization.
Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: Rigid-body fitting in Chimera was followed by manual building in Coot and refinement using Phenix real-space refine and Refmac-Servalcat.
Atomic model buildingPDB-ID: 7N00

7n00


Accession code: 7N00 / Source name: PDB / Type: experimental model

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