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- EMDB-51087: Cryo-EM structure of a 2:1 ALK:ALKAL2 complex obtained after re-p... -

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Entry
Database: EMDB / ID: EMD-51087
TitleCryo-EM structure of a 2:1 ALK:ALKAL2 complex obtained after re-processing of EMPIAR-10930 data
Map dataRegularly sharpened map of the 2:1 ALK-ALKAL2 complex, obtained after re-processing of EMPIAR-10930 data.
Sample
  • Complex: Cryo-EM structure of a 2:1 ALK:ALKAL2 complex obtained after re-processing of EMPIAR-10930 data
    • Protein or peptide: ALK tyrosine kinase receptor
    • Protein or peptide: ALK and LTK ligand 2
KeywordsComplex / receptor tyrosine kinase / CYTOKINE
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 / transmembrane receptor protein tyrosine kinase activator activity / ALK mutants bind TKIs / swimming behavior / Signaling by LTK / regulation of neuron differentiation / positive regulation of dendrite development / Signaling by ALK / phosphorylation / response to stress / adult behavior / neuron development / negative regulation of lipid catabolic process / peptidyl-tyrosine autophosphorylation / energy homeostasis / transmembrane receptor protein tyrosine kinase activity / cell surface receptor protein tyrosine kinase signaling pathway / hippocampus development / cytokine activity / placental growth factor receptor activity / insulin receptor activity / vascular endothelial growth factor receptor activity / hepatocyte growth factor receptor activity / macrophage colony-stimulating factor receptor activity / platelet-derived growth factor alpha-receptor activity / platelet-derived growth factor beta-receptor activity / stem cell factor receptor activity / boss receptor activity / protein tyrosine kinase collagen receptor activity / brain-derived neurotrophic factor receptor activity / transmembrane-ephrin receptor activity / GPI-linked ephrin receptor activity / epidermal growth factor receptor activity / fibroblast growth factor receptor activity / insulin-like growth factor receptor activity / receptor protein-tyrosine kinase / positive regulation of neuron projection development / receptor tyrosine kinase binding / positive regulation of NF-kappaB transcription factor activity / 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 / receptor complex / positive regulation of ERK1 and ERK2 cascade / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsFelix J / De Munck S / Bazan JF / Savvides SN
Funding support Belgium, 2 items
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, 2024-
Header (metadata) releaseMar 12, 2025-
Map releaseMar 12, 2025-
UpdateApr 23, 2025-
Current statusApr 23, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51087.png

Downloads & links

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Map

FileDownload / File: emd_51087.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRegularly sharpened map of the 2:1 ALK-ALKAL2 complex, obtained after re-processing of EMPIAR-10930 data.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 211.456 Å		0.83 Å/pix.
x 256 pix.
= 211.456 Å		0.83 Å/pix.
x 256 pix.
= 211.456 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.826 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.057428874 - 0.10045845
Average (Standard dev.)0.000117186944 (±0.0022986536)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.456 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_51087_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: DeepEMhancer sharpened map of the 2:1 ALK-ALKAL2 complex,...

Fileemd_51087_additional_1.map
AnnotationDeepEMhancer sharpened map of the 2:1 ALK-ALKAL2 complex, obtained after re-processing of EMPIAR-10930 data.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of the 2:1 ALK-ALKAL2 complex,...

Fileemd_51087_half_map_1.map
AnnotationHalf map 1 of the 2:1 ALK-ALKAL2 complex, obtained after re-processing of EMPIAR-10930 data.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of the 2:1 ALK-ALKAL2 complex,...

Fileemd_51087_half_map_2.map
AnnotationHalf map 2 of the 2:1 ALK-ALKAL2 complex, obtained after re-processing of EMPIAR-10930 data.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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

EntireName: Cryo-EM structure of a 2:1 ALK:ALKAL2 complex obtained after re-processing of EMPIAR-10930 data
Components
  • Complex: Cryo-EM structure of a 2:1 ALK:ALKAL2 complex obtained after re-processing of EMPIAR-10930 data
    • Protein or peptide: ALK tyrosine kinase receptor
    • Protein or peptide: ALK and LTK ligand 2

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Supramolecule #1: Cryo-EM structure of a 2:1 ALK:ALKAL2 complex obtained after re-p...

SupramoleculeName: Cryo-EM structure of a 2:1 ALK:ALKAL2 complex obtained after re-processing of EMPIAR-10930 data
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 86 KDa

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Macromolecule #1: ALK tyrosine kinase receptor

MacromoleculeName: ALK tyrosine kinase receptor / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: receptor protein-tyrosine kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.377559 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GTAPKSRNLF ERNPNKELKP GENSPRQTPI FDPTVHWLFT TCGASGPHGP TQAQCNNAYQ NSNLSVEVGS EGPLKGIQIW KVPATDTYS ISGYGAAGGK GGKNTMMRSH GVSVLGIFNL EKDDMLYILV GQQGEDACPS TNQLIQKVCI GENNVIEEEI R VNRSVHEW ...String:
GTAPKSRNLF ERNPNKELKP GENSPRQTPI FDPTVHWLFT TCGASGPHGP TQAQCNNAYQ NSNLSVEVGS EGPLKGIQIW KVPATDTYS ISGYGAAGGK GGKNTMMRSH GVSVLGIFNL EKDDMLYILV GQQGEDACPS TNQLIQKVCI GENNVIEEEI R VNRSVHEW AGGGGGGGGA TYVFKMKDGV PVPLIIAAGG GGRAYGAKTD TFHPERLENN SSVLGLNGNS GAAGGGGGWN DN TSLLWAG KSLQEGATGG HSCPQAMKKW GWETRGGFGG GGGGCSSGGG GGGYIGGNAA SNNDPEMDGE DGVSFISPLG ILY TPALKV MEGHGEVNIK HYLNCSHCEV DECHMDPESH KVICFCDHGT VLAEDGVSCI VSP

UniProtKB: ALK tyrosine kinase receptor

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Macromolecule #2: ALK and LTK ligand 2

MacromoleculeName: ALK and LTK ligand 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.640013 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GAEPREPADG QALLRLVVEL VQELRKHHSA EHKGLQLLGR DYALGRAEAA GLGPSPEQRV EIVPRDLRMK DKFLKHLTGP LYFSPKCSK HFHRLYHNTR DCTIPAYYKR CARLLTRLAV SPVCMEDKQ

UniProtKB: ALK and LTK ligand 2

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.4
Component:
ConcentrationName
15.0 mMHEPES
150.0 mMNaCl
3.0 %PEG4000
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI TITAN KRIOS
TemperatureMin: 96.0 K / Max: 98.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
DetailsSerialEM coma-free alignment
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 13618 / Average exposure time: 4.2 sec. / Average electron dose: 81.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 18053705
Startup modelType of model: OTHER / Details: cryoSPARC Ab-Initio Reconstruction.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5)
Details: Final 3D refinement was performed in Relion v5 with enabled Blush Regularization.
Number images used: 142986
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 4.5) / Details: Stochastic gradient descent (SGD).
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 4.5)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7n00


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsRigid-body fitting in Chimera was followed by manual building in Coot and refinement using Phenix real-space refine and Refmac-Servalcat.
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-9g5i:
Cryo-EM structure of a 2:1 ALK:ALKAL2 complex obtained after re-processing of EMPIAR-10930 data

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