+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-40587 | |||||||||
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Title | CryoEM reconstruction of full-length Btk (class 3) | |||||||||
Map data | sharpened refined map | |||||||||
Sample |
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Keywords | Bruton's Tyrosine Kinase / ATP-binding / protein phosphorylation / TRANSFERASE | |||||||||
Function / homology | Function and homology information negative regulation of B cell activation / G beta:gamma signalling through BTK / RHO GTPases Activate WASPs and WAVEs / G alpha (q) signalling events / FCERI mediated Ca+2 mobilization / G alpha (12/13) signalling events / negative regulation of leukocyte proliferation / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / Regulation of actin dynamics for phagocytic cup formation / proteoglycan catabolic process ...negative regulation of B cell activation / G beta:gamma signalling through BTK / RHO GTPases Activate WASPs and WAVEs / G alpha (q) signalling events / FCERI mediated Ca+2 mobilization / G alpha (12/13) signalling events / negative regulation of leukocyte proliferation / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / Regulation of actin dynamics for phagocytic cup formation / proteoglycan catabolic process / monocyte proliferation / positive regulation of interleukin-17A production / eosinophil homeostasis / positive regulation of type III hypersensitivity / B cell affinity maturation / positive regulation of synoviocyte proliferation / histamine secretion by mast cell / neutrophil homeostasis / cellular response to molecule of fungal origin / positive regulation of type I hypersensitivity / cellular response to interleukin-7 / DAP12 signaling / negative regulation of cytokine production / positive regulation of immunoglobulin production / positive regulation of NLRP3 inflammasome complex assembly / phospholipase activator activity / negative regulation of interleukin-10 production / negative regulation of B cell proliferation / phosphatidylinositol-3,4,5-trisphosphate binding / phospholipase binding / positive regulation of phagocytosis / positive regulation of B cell proliferation / cell maturation / B cell receptor signaling pathway / non-specific protein-tyrosine kinase / non-membrane spanning protein tyrosine kinase activity / positive regulation of interleukin-6 production / cellular response to reactive oxygen species / positive regulation of tumor necrosis factor production / T cell receptor signaling pathway / protein tyrosine kinase activity / cytoplasmic vesicle / adaptive immune response / response to lipopolysaccharide / intracellular signal transduction / membrane raft / innate immune response / apoptotic process / perinuclear region of cytoplasm / ATP binding / identical protein binding / nucleus / metal ion binding / plasma membrane / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Mus musculus (house mouse) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 5.56 Å | |||||||||
Authors | Lin DY / Andreotti AH / Juneja P | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Elife / Year: 2024 Title: Conformational heterogeneity of the BTK PHTH domain drives multiple regulatory states. Authors: David Yin-Wei Lin / Lauren E Kueffer / Puneet Juneja / Thomas E Wales / John R Engen / Amy H Andreotti / Abstract: Full-length Bruton's tyrosine kinase (BTK) has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely ...Full-length Bruton's tyrosine kinase (BTK) has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely how the BTK N-terminal domains (the Pleckstrin homology/Tec homology [PHTH] domain and proline-rich regions [PRR] contain linker) contribute to BTK regulation remains unclear. We have produced crystals of full-length BTK for the first time but despite efforts to stabilize the autoinhibited state, the diffraction data still reveal only the SH3-SH2-kinase core with no electron density visible for the PHTH-PRR segment. Cryo-electron microscopy (cryoEM) data of full-length BTK, on the other hand, provide the first view of the PHTH domain within full-length BTK. CryoEM reconstructions support conformational heterogeneity in the PHTH-PRR region wherein the globular PHTH domain adopts a range of states arrayed around the autoinhibited SH3-SH2-kinase core. On the way to activation, disassembly of the SH3-SH2-kinase core opens a new autoinhibitory site on the kinase domain for PHTH domain binding that is ultimately released upon interaction of PHTH with phosphatidylinositol (3,4,5)-trisphosphate. Membrane-induced dimerization activates BTK and we present here a crystal structure of an activation loop swapped BTK kinase domain dimer that likely represents the conformational state leading to trans-autophosphorylation. Together, these data provide the first structural elucidation of full-length BTK and allow a deeper understanding of allosteric control over the BTK kinase domain during distinct stages of activation. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_40587.map.gz | 206.9 MB | EMDB map data format | |
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Header (meta data) | emd-40587-v30.xml emd-40587.xml | 18.4 KB 18.4 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_40587_fsc.xml | 12.7 KB | Display | FSC data file |
Images | emd_40587.png | 70.4 KB | ||
Filedesc metadata | emd-40587.cif.gz | 5.9 KB | ||
Others | emd_40587_half_map_1.map.gz emd_40587_half_map_2.map.gz | 203.8 MB 203.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-40587 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-40587 | HTTPS FTP |
-Validation report
Summary document | emd_40587_validation.pdf.gz | 988.5 KB | Display | EMDB validaton report |
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Full document | emd_40587_full_validation.pdf.gz | 988.1 KB | Display | |
Data in XML | emd_40587_validation.xml.gz | 21.2 KB | Display | |
Data in CIF | emd_40587_validation.cif.gz | 27.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-40587 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-40587 | HTTPS FTP |
-Related structure data
Related structure data | 8gmbC 8s93C 8s9fC 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_40587.map.gz / Format: CCP4 / Size: 219.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | sharpened refined map | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.45315 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: half map B
File | emd_40587_half_map_1.map | ||||||||||||
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Annotation | half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map A
File | emd_40587_half_map_2.map | ||||||||||||
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Annotation | half map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Full-length Bruton's Tyrosine Kinase
Entire | Name: Full-length Bruton's Tyrosine Kinase |
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Components |
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-Supramolecule #1: Full-length Bruton's Tyrosine Kinase
Supramolecule | Name: Full-length Bruton's Tyrosine Kinase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Mus musculus (house mouse) |
-Macromolecule #1: Full-length Bruton's Tyrosine Kinase
Macromolecule | Name: Full-length Bruton's Tyrosine Kinase / type: protein_or_peptide / ID: 1 / Details: Mouse full-length Btk with inhibitory mutations / Enantiomer: LEVO / EC number: non-specific protein-tyrosine kinase |
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Source (natural) | Organism: Mus musculus (house mouse) |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MAAVILESIF LKRSQQKKKT SPLNFKKRLF LLTVHKLSYY EYDFERGRRG SKKGSIDVEK ITCVETVIP EKNPPPERQI PRRGEESSEM EQISIIERFP YPFQVVYDEG PLYVFSPTEE L RKRWIHQL KNVIRYNSDL VQKYHPCFWI DGQYLCCSQT AKNAMGCQIL ...String: MAAVILESIF LKRSQQKKKT SPLNFKKRLF LLTVHKLSYY EYDFERGRRG SKKGSIDVEK ITCVETVIP EKNPPPERQI PRRGEESSEM EQISIIERFP YPFQVVYDEG PLYVFSPTEE L RKRWIHQL KNVIRYNSDL VQKYHPCFWI DGQYLCCSQT AKNAMGCQIL ENRNGSLKPG SS HRKTKKP LPPTPEEDQI LKKPLPPEPT AAPISTTELK KVVALYDYMP MNANDLQLRK GEE YFILEE SNLPWWRARD KNGQEGYIPS NYITEAEDSI EMYEWYSKHM TRSQAEQLLK QAGA AGGFI VRDSSKAGKY TVSVFAKSTG EPQGVIRHYV VCSTPQSQYY LAEKHLFSTI PELIN YHQH NSAGLISRLK YPVSKQNKNP PPPPGFGYGS WEIDPKDLTF LKELGTGQFG VVKYGK WRG QYDVAIRMIR EGSMSEDEFI EEAKVMMNLS HEKLVQLYGV CTKQRPIFII TEYMANG CL LNYLREMRHR FQTQQLLEMC KDVCEAMEYL ESKQFLHRDL AARNCLVNDQ GVVKVSDF G MTRYVLDDEY TSSTGSKFPV KWASPEVLMY SKFSSKSDIW AFGVLMWEIY SLGKMPYER FTNSETAEHI AQGLRLPRPH LASERVYTIM YSCWHEKADE RPSFKILLSN ILDVMDEES UniProtKB: Tyrosine-protein kinase BTK |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.5 mg/mL | ||||||||
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Buffer | pH: 8 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. | ||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV | ||||||||
Details | The sample was monodisperse. |
-Electron microscopy
Microscope | TFS GLACIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 1 / Number real images: 755 / Average exposure time: 2.0 sec. / Average electron dose: 59.0 e/Å2 Details: Images were collected in Super-resolution made at 50 frames per second |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 45000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |