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Open data
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Basic information
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Title | Cryo-EM structure of LKB1-STRADalpha-MO25alpha heterocomplex | |||||||||
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![]() | serine/threonine kinase / pseudokinase / complex / TRANSFERASE | |||||||||
Function / homology | ![]() 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 / regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction ...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 / regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / serine/threonine protein kinase complex / cellular hypotonic response / response to lipid / 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 / establishment of cell polarity / cellular response to UV-B / regulation of dendrite morphogenesis / response to ionizing radiation / G1 to G0 transition / activation of protein kinase activity / protein kinase activator activity / positive regulation of transforming growth factor beta receptor signaling pathway / 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 dephosphorylation / protein serine/threonine kinase activator activity / axonogenesis / positive regulation of peptidyl-threonine phosphorylation / regulation of signal transduction by p53 class mediator / response to activity / regulation of cell growth / kinase binding / peptidyl-threonine phosphorylation / negative regulation of canonical Wnt signaling pathway / positive regulation of protein serine/threonine kinase activity / negative regulation of cell growth / p53 binding / Z disc / autophagy / positive regulation of protein localization to nucleus / glucose homeostasis / peptidyl-serine phosphorylation / T cell receptor signaling pathway / spermatogenesis / Regulation of TP53 Activity through Phosphorylation / secretory granule lumen / protein autophosphorylation / ficolin-1-rich granule lumen / regulation of cell cycle / non-specific serine/threonine protein kinase / intracellular signal transduction / protein phosphorylation / negative regulation of cell population proliferation / 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 | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.86 Å | |||||||||
![]() | Chan LM / Courteau BJ / Verba KA | |||||||||
Funding support | 1 items
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![]() | ![]() 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 / Hev 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 / Hev 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 energies, 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 of a 115 kDa asymmetric protein complex, proving Alpine's 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, bringing smaller sized particles within the scope of cryo-EM. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 59.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 23.2 KB 23.2 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 8.8 KB | Display | ![]() |
Images | ![]() | 69.5 KB | ||
Filedesc metadata | ![]() | 7.1 KB | ||
Others | ![]() ![]() ![]() | 31.9 MB 59.5 MB 59.5 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 889.1 KB | Display | ![]() |
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Full document | ![]() | 888.7 KB | Display | |
Data in XML | ![]() | 16.4 KB | Display | |
Data in CIF | ![]() | 21.3 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8vsuMC C: citing same article ( M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 0.835 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: #1
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Density Histograms |
-Half map: #2
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Projections & Slices |
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Density Histograms |
-Half map: #1
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Density Histograms |
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Sample components
-Entire : Heterotrimeric complex of serine/threonine kinase LKB1 with psued...
Entire | Name: Heterotrimeric complex of serine/threonine kinase LKB1 with psuedokinase STRADa and scaffolding MO25a |
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Components |
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-Supramolecule #1: Heterotrimeric complex of serine/threonine kinase LKB1 with psued...
Supramolecule | Name: Heterotrimeric complex of serine/threonine kinase LKB1 with psuedokinase STRADa and scaffolding MO25a type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 140.5 KDa |
-Macromolecule #1: Calcium-binding protein 39
Macromolecule | Name: Calcium-binding protein 39 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 41.997109 KDa |
Recombinant expression | Organism: ![]() |
Sequence | String: 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 |
-Macromolecule #2: Serine/threonine-protein kinase STK11
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 50.649785 KDa |
Recombinant expression | Organism: ![]() |
Sequence | String: 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 |
-Macromolecule #3: Isoform 3 of STE20-related kinase adapter protein alpha
Macromolecule | Name: Isoform 3 of STE20-related kinase adapter protein alpha type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 47.861875 KDa |
Recombinant expression | Organism: ![]() |
Sequence | String: 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 |
-Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: ADP |
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Molecular weight | Theoretical: 427.201 Da |
Chemical component information | ![]() ChemComp-ADP: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.6 Component:
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Grid | Model: 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 | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III |
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Electron microscopy
Microscope | TFS KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 1293 / Average electron dose: 45.8 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 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 stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Refinement | Protocol: RIGID BODY FIT |
Output model | ![]() PDB-8vsu: |
-Atomic model buiding 2
Refinement | Protocol: FLEXIBLE FIT |
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Output model | ![]() PDB-8vsu: |