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- EMDB-43506: Cryo-EM structure of LKB1-STRADalpha-MO25alpha heterocomplex -

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Basic information

Entry
Database: EMDB / ID: EMD-43506
TitleCryo-EM structure of LKB1-STRADalpha-MO25alpha heterocomplex
Map data
Sample
  • Complex: Heterotrimeric complex of serine/threonine kinase LKB1 with psuedokinase STRADa and scaffolding MO25a
    • Protein or peptide: Calcium-binding protein 39
    • Protein or peptide: Serine/threonine-protein kinase STK11
    • Protein or peptide: Isoform 3 of STE20-related kinase adapter protein alpha
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Keywordsserine/threonine kinase / pseudokinase / complex / TRANSFERASE
Function / homology
Function and homology information


positive regulation of vesicle transport along microtubule / intracellular protein-containing complex / Golgi localization / LRR domain binding / negative regulation of potassium ion transmembrane transporter activity / AMPK inhibits chREBP transcriptional activation activity / negative regulation of potassium ion transmembrane transport / dendrite extension / negative regulation of epithelial cell proliferation involved in prostate gland development / serine/threonine protein kinase complex ...positive regulation of vesicle transport along microtubule / intracellular protein-containing complex / Golgi localization / LRR domain binding / negative regulation of potassium ion transmembrane transporter activity / AMPK inhibits chREBP transcriptional activation activity / negative regulation of potassium ion transmembrane transport / dendrite extension / negative regulation of epithelial cell proliferation involved in prostate gland development / serine/threonine protein kinase complex / cellular hypotonic response / tissue homeostasis / epithelial cell proliferation involved in prostate gland development / response to thyroid hormone / Energy dependent regulation of mTOR by LKB1-AMPK / positive thymic T cell selection / vasculature development / regulation of Wnt signaling pathway / anoikis / response to glucagon / negative regulation of cold-induced thermogenesis / FOXO-mediated transcription of cell death genes / positive regulation of axonogenesis / regulation of dendrite morphogenesis / establishment of cell polarity / G1 to G0 transition / response to ionizing radiation / cellular response to UV-B / response to lipid / activation of protein kinase activity / positive regulation of transforming growth factor beta receptor signaling pathway / protein kinase activator activity / regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / intrinsic apoptotic signaling pathway by p53 class mediator / protein localization to nucleus / positive regulation of autophagy / positive regulation of gluconeogenesis / negative regulation of TORC1 signaling / protein export from nucleus / protein serine/threonine kinase activator activity / protein dephosphorylation / axonogenesis / positive regulation of peptidyl-threonine phosphorylation / regulation of signal transduction by p53 class mediator / response to activity / regulation of cell growth / peptidyl-threonine phosphorylation / negative regulation of canonical Wnt signaling pathway / positive regulation of protein serine/threonine kinase activity / negative regulation of cell growth / autophagy / kinase binding / Z disc / positive regulation of protein localization to nucleus / p53 binding / glucose homeostasis / T cell receptor signaling pathway / spermatogenesis / peptidyl-serine phosphorylation / secretory granule lumen / Regulation of TP53 Activity through Phosphorylation / ficolin-1-rich granule lumen / protein autophosphorylation / non-specific serine/threonine protein kinase / regulation of cell cycle / intracellular signal transduction / negative regulation of cell population proliferation / protein phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / DNA damage response / Neutrophil degranulation / protein-containing complex binding / magnesium ion binding / signal transduction / mitochondrion / extracellular exosome / extracellular region / nucleoplasm / ATP binding / membrane / nucleus / cytoplasm / cytosol
Similarity search - Function
Serine/Threonine kinase LKB1, catalytic domain / : / Mo25-like / Mo25-like / Armadillo-like helical / Armadillo-type fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain ...Serine/Threonine kinase LKB1, catalytic domain / : / Mo25-like / Mo25-like / Armadillo-like helical / Armadillo-type fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / 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
Serine/threonine-protein kinase STK11 / STE20-related kinase adapter protein alpha / Calcium-binding protein 39
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsChan LM / Courteau BJ / Verba KA
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: J Struct Biol / Year: 2024
Title: High-resolution single-particle imaging at 100-200 keV with the Gatan Alpine direct electron detector.
Authors: Lieza M Chan / Brandon J Courteau / Allison Maker / Mengyu Wu / Benjamin Basanta / Hevatib Mehmood / David Bulkley / David Joyce / Brian C Lee / Stephen Mick / Cory Czarnik / Sahil Gulati / ...Authors: Lieza M Chan / Brandon J Courteau / Allison Maker / Mengyu Wu / Benjamin Basanta / Hevatib Mehmood / David Bulkley / David Joyce / Brian C Lee / Stephen Mick / Cory Czarnik / Sahil Gulati / Gabriel C Lander / Kliment A Verba /
Abstract: Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments ...Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imaging at 100 keV, energies for which the current detectors have not been optimized. In this study, we evaluated the Gatan Alpine detector, designed for operation at 100 and 200 keV. Compared to the Gatan K3, Alpine demonstrated a significant DQE improvement at these voltages, specifically a ∼ 4-fold improvement at Nyquist at 100 keV. In single-particle cryo-EM experiments, Alpine datasets yielded better than 2 Å resolution reconstructions of apoferritin at 120 and 200 keV on a ThermoFisher Scientific (TFS) Glacios microscope fitted with a non-standard SP-Twin lens. We also achieved a ∼ 3.2 Å resolution reconstruction for a 115 kDa asymmetric protein complex, proving its effectiveness with complex biological samples. In-depth analysis revealed that Alpine reconstructions are comparable to K3 reconstructions at 200 keV, and remarkably, reconstruction from Alpine at 120 keV on a TFS Glacios surpassed all but the 300 keV data from a TFS Titan Krios with GIF/K3. Additionally, we show Alpine's capability for high-resolution data acquisition and screening on lower-end systems by obtaining ∼ 3 Å resolution reconstructions of apoferritin and aldolase at 100 keV and detailed 2D averages of a 55 kDa sample using a side-entry cryo holder. Overall, we show that Gatan Alpine performs well with the standard 200 keV imaging systems and may potentially capture the benefits of lower accelerating voltages, possibly bringing smaller sized particles within the scope of cryo-EM.
History
DepositionJan 24, 2024-
Header (metadata) releaseJul 10, 2024-
Map releaseJul 10, 2024-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: RCSB / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_43506.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.835 Å
Density
Contour LevelBy AUTHOR: 0.628
Minimum - Maximum-2.21325 - 4.1446104
Average (Standard dev.)-0.00062040903 (±0.10249217)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 213.76 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Heterotrimeric complex of serine/threonine kinase LKB1 with psued...

EntireName: Heterotrimeric complex of serine/threonine kinase LKB1 with psuedokinase STRADa and scaffolding MO25a
Components
  • Complex: Heterotrimeric complex of serine/threonine kinase LKB1 with psuedokinase STRADa and scaffolding MO25a
    • Protein or peptide: Calcium-binding protein 39
    • Protein or peptide: Serine/threonine-protein kinase STK11
    • Protein or peptide: Isoform 3 of STE20-related kinase adapter protein alpha
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Heterotrimeric complex of serine/threonine kinase LKB1 with psued...

SupramoleculeName: Heterotrimeric complex of serine/threonine kinase LKB1 with psuedokinase STRADa and scaffolding MO25a
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 140.5 KDa

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Macromolecule #1: Calcium-binding protein 39

MacromoleculeName: Calcium-binding protein 39 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.997109 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MPFPFGKSHK SPADIVKNLK ESMAVLEKQD ISDKKAEKAT EEVSKNLVAM KEILYGTNEK EPQTEAVAQL AQELYNSGLL STLVADLQL IDFEGKKDVA QIFNNILRRQ IGTRTPTVEY ICTQQNILFM LLKGYESPEI ALNCGIMLRE CIRHEPLAKI I LWSEQFYD ...String:
MPFPFGKSHK SPADIVKNLK ESMAVLEKQD ISDKKAEKAT EEVSKNLVAM KEILYGTNEK EPQTEAVAQL AQELYNSGLL STLVADLQL IDFEGKKDVA QIFNNILRRQ IGTRTPTVEY ICTQQNILFM LLKGYESPEI ALNCGIMLRE CIRHEPLAKI I LWSEQFYD FFRYVEMSTF DIASDAFATF KDLLTRHKLL SAEFLEQHYD RFFSEYEKLL HSENYVTKRQ SLKLLGELLL DR HNFTIMT KYISKPENLK LMMNLLRDKS RNIQFEAFHV FKVFVANPNK TQPILDILLK NQAKLIEFLS KFQNDRTEDE QFN DEKTYL VKQIRDLKRP AQQEAGSGAT NFSLLKQAGD VEENPG

UniProtKB: Calcium-binding protein 39

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Macromolecule #2: Serine/threonine-protein kinase STK11

MacromoleculeName: Serine/threonine-protein kinase STK11 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 50.649785 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: PDYKDDDDKE NLYFQGMEVV DPQQLGMFTE GELMSVGMDT FIHRIDSTEV IYQPRRKRAK LIGKYLMGDL LGEGSYGKVK EVLDSETLC RRAVKILKKK KLRRIPNGEA NVKKEIQLLR RLRHKNVIQL VDVLYNEEKQ KMYMVMEYCV CGMQEMLDSV P EKRFPVCQ ...String:
PDYKDDDDKE NLYFQGMEVV DPQQLGMFTE GELMSVGMDT FIHRIDSTEV IYQPRRKRAK LIGKYLMGDL LGEGSYGKVK EVLDSETLC RRAVKILKKK KLRRIPNGEA NVKKEIQLLR RLRHKNVIQL VDVLYNEEKQ KMYMVMEYCV CGMQEMLDSV P EKRFPVCQ AHGYFCQLID GLEYLHSQGI VHKDIKPGNL LLTTGGTLKI SDLGVAEALH PFAADDTCRT SQGSPAFQPP EI ANGLDTF SGFKVDIWSA GVTLYNITTG LYPFEGDNIY KLFENIGKGS YAIPGDCGPP LSDLLKGMLE YEPAKRFSIR QIR QHSWFR KKHPPAEAPV PIPPSPDTKD RWRSMTVVPY LEDLHGADED EDLFDIEDDI IYTQDFTVPG QVPEEEASHN GQRR GLPKA VCMNGTEAAQ LSTKSRAEGR APNPARKACS ASSKIRRLSA CKQQ

UniProtKB: Serine/threonine-protein kinase STK11

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Macromolecule #3: Isoform 3 of STE20-related kinase adapter protein alpha

MacromoleculeName: Isoform 3 of STE20-related kinase adapter protein alpha
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 47.861875 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: PHHHHHHENL YFQGMSFLTN DASSESIASF SKQEVMSSFL PEGGCYELLT VIGKGFEDLM TVNLARYKPT GEYVTVRRIN LEACSNEMV TFLQGELHVS KLFNHPNIVP YRATFIADNE LWVVTSFMAY GSAKDLICTH FMDGMNELAI AYILQGVLKA L DYIHHMGY ...String:
PHHHHHHENL YFQGMSFLTN DASSESIASF SKQEVMSSFL PEGGCYELLT VIGKGFEDLM TVNLARYKPT GEYVTVRRIN LEACSNEMV TFLQGELHVS KLFNHPNIVP YRATFIADNE LWVVTSFMAY GSAKDLICTH FMDGMNELAI AYILQGVLKA L DYIHHMGY VHRSVKASHI LISVDGKVYL SGLRSNLSMI SHGQRQRVVH DFPKYSVKVL PWLSPEVLQQ NLQGYDAKSD IY SVGITAC ELANGHVPFK DMPATQMLLE KLNGTVPCLL DTSTIPAEEL TMSPSRSVAN SGLSDSLTTS TPRPSNGDSP SHP YHRTFS PHFHHFVEQC LQRNPDARPS ASTLLNHSFF KQIKRRASEA LPELLRPVTP ITNFEGSQSQ DHSGIFGLVT NLEE LEVDD WEFGSGATNF SLLKQAGDVE ENPG

UniProtKB: STE20-related kinase adapter protein alpha

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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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

BufferpH: 7.6
Component:
ConcentrationNameFormula
25.0 mMTris HCl
350.0 mMsodium chlorideNaCl
5.0 %glycerol
0.5 mMDTT
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.033 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 1293 / Average electron dose: 45.8 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: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 979927
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2wtk

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 124780
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

2wtk


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-8vsu:
Cryo-EM structure of LKB1-STRADalpha-MO25alpha heterocomplex

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Atomic model buiding 2

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-8vsu:
Cryo-EM structure of LKB1-STRADalpha-MO25alpha heterocomplex

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