EMDB-40586: CryoEM reconstruction of full-length Btk (class 1)

Basic information

Entry

Database: EMDB / ID: EMD-40586

• Title: CryoEM reconstruction of full-length Btk (class 1)
• Map data: Sharpened map

Sample

• Complex: Full-length Bruton's Tyrosine Kinase
  • Protein or peptide: Full-length Bruton's Tyrosine Kinase

• Keywords: Bruton's Tyrosine Kinase / ATP-binding / protein phosphorylation / TRANSFERASE

Function / homology

Function:

negative regulation of B cell activation / G beta:gamma signalling through BTK / RHO GTPases Activate WASPs and WAVEs / FCERI mediated Ca+2 mobilization / G alpha (q) signalling events / G alpha (12/13) signalling events / 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 / histamine secretion by mast cell / positive regulation of synoviocyte proliferation / negative regulation of leukocyte proliferation / 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 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 immunoglobulin production / positive regulation of phagocytosis / positive regulation of B cell proliferation / cell maturation / response to organic substance / B cell receptor signaling pathway / non-specific protein-tyrosine kinase / non-membrane spanning protein tyrosine kinase activity / cellular response to reactive oxygen species / positive regulation of interleukin-6 production / positive regulation of tumor necrosis factor production / T cell receptor signaling pathway / cytoplasmic vesicle / protein tyrosine kinase activity / response to lipopolysaccharide / adaptive immune response / intracellular signal transduction / membrane raft / phosphorylation / innate immune response / apoptotic process / perinuclear region of cytoplasm / ATP binding / identical protein binding / metal ion binding / nucleus / plasma membrane / cytosol / cytoplasm

Domain/homology:

Tyrosine-protein kinase BTK, SH3 domain / Zinc finger, Btk motif / BTK motif / Zinc finger Btk-type profile. / Bruton's tyrosine kinase Cys-rich motif / PH domain / PH domain profile. / Pleckstrin homology domain. / Pleckstrin homology domain / SH3 domain / SH2 domain / Src homology 2 (SH2) domain profile. / Src homology 2 domains / SH2 domain / Src homology 3 domains / SH2 domain superfamily / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Tyrosine-protein kinase, catalytic domain / Tyrosine kinase, catalytic domain / Tyrosine protein kinases specific active-site signature. / PH-like domain superfamily / Tyrosine-protein kinase, active site / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, 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

Component:

Tyrosine-protein kinase BTK

• Biological species: Mus musculus (house mouse)
• Method: single particle reconstruction / cryo EM / Resolution: 6.36 Å
• Authors: Lin DY / Andreotti AH / Juneja P

Funding support

United States, 1 items

Organization	Grant number	Country
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)		United States

• 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 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

Deposition	Apr 25, 2023	-
Header (metadata) release	Aug 16, 2023	-
Map release	Aug 16, 2023	-
Update	Jan 31, 2024	-
Current status	Jan 31, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_40586.map.gz / Format: CCP4 / Size: 219.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Sharpened map
• Voxel size: X=Y=Z: 0.45315 Å

Density

Contour Level	By AUTHOR: 0.04
Minimum - Maximum	-0.30817124 - 0.5354159
Average (Standard dev.)	0.00020685597 (±0.0075367843)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 386 386 386 Spacing 386 386 386
Cell	A=B=C: 174.91591 Å α=β=γ: 90.0 °

Supplemental data

Half map: half map A

• File: emd_40586_half_map_1.map
• Annotation: half map A

Half map: half map B

• File: emd_40586_half_map_2.map
• Annotation: half map B

Sample components

Entire : Full-length Bruton's Tyrosine Kinase

• Entire: Name: Full-length Bruton's Tyrosine Kinase

Components

• Complex: Full-length Bruton's Tyrosine Kinase
  • Protein or peptide: Full-length Bruton's Tyrosine Kinase

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
• 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
• 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 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
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.5 mg/mL

Buffer

pH: 8
Component:

Concentration	Name
20.0 mM	Tris(hydroxymethyl)aminomethane
150.0 mM	sodium chloride
1.0 mM	Dithiothreitol

• 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
• 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 FIELDBright-field microscopy / 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
• 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/Ų; Details: Images were collected in Super-resolution made at 50 frames per second

Image processing

• Particle selection: Number selected: 2268597
• Startup model: Type of model: INSILICO MODEL
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.2)
• Final 3D classification: Number classes: 4 / Avg.num./class: 16000 / Software - Name: cryoSPARC (ver. 4.1.2)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.2)
• Final reconstruction: Number classes used: 1 / Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.36 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1.2) / Number images used: 19275