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- PDB-7dac: Human RIPK3 amyloid fibril revealed by solid-state NMR -

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Basic information

Entry
Database: PDB / ID: 7dac
TitleHuman RIPK3 amyloid fibril revealed by solid-state NMR
ComponentsReceptor-interacting serine/threonine-protein kinase 3
KeywordsPROTEIN FIBRIL / programmed necrosis / amyloid
Function / homology
Function and homology information


regulation of activation-induced cell death of T cells / regulation of CD8-positive, alpha-beta cytotoxic T cell extravasation / execution phase of necroptosis / regulation of T cell mediated cytotoxicity / regulation of adaptive immune response / regulation of activated T cell proliferation / positive regulation of phosphatase activity / regulation of type II interferon production / TRIF-mediated programmed cell death / TLR3-mediated TICAM1-dependent programmed cell death ...regulation of activation-induced cell death of T cells / regulation of CD8-positive, alpha-beta cytotoxic T cell extravasation / execution phase of necroptosis / regulation of T cell mediated cytotoxicity / regulation of adaptive immune response / regulation of activated T cell proliferation / positive regulation of phosphatase activity / regulation of type II interferon production / TRIF-mediated programmed cell death / TLR3-mediated TICAM1-dependent programmed cell death / Microbial modulation of RIPK1-mediated regulated necrosis / programmed necrotic cell death / SARS-CoV-1-mediated effects on programmed cell death / necroptotic signaling pathway / positive regulation of necroptotic process / RIP-mediated NFkB activation via ZBP1 / TRP channels / RIPK1-mediated regulated necrosis / activation of protein kinase activity / non-canonical NF-kappaB signal transduction / T cell homeostasis / necroptotic process / lymph node development / positive regulation of intrinsic apoptotic signaling pathway / spleen development / reactive oxygen species metabolic process / TICAM1, RIP1-mediated IKK complex recruitment / IKK complex recruitment mediated by RIP1 / thymus development / apoptotic signaling pathway / protein modification process / Regulation of necroptotic cell death / cellular response to hydrogen peroxide / positive regulation of reactive oxygen species metabolic process / SARS-CoV-1 activates/modulates innate immune responses / positive regulation of NF-kappaB transcription factor activity / T cell differentiation in thymus / regulation of apoptotic process / defense response to virus / amyloid fibril formation / protein autophosphorylation / transcription coactivator activity / non-specific serine/threonine protein kinase / protein kinase activity / protein serine kinase activity / protein serine/threonine kinase activity / protein-containing complex binding / signal transduction / protein-containing complex / ATP binding / identical protein binding / nucleus / cytoplasm / cytosol
Similarity search - Function
RHIM domain / RIP homotypic interaction motif / 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
Receptor-interacting serine/threonine-protein kinase 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLID-STATE NMR / simulated annealing
AuthorsWu, X.L. / Zhang, J. / Dong, X.Q. / Liu, J. / Li, B. / Hu, H. / Wang, J. / Wang, H.Y. / Lu, J.X.
Funding support China, 1items
OrganizationGrant numberCountry
Other governmentMinistry of Science and Technology (China) 2017YFA0504804 China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: The structure of a minimum amyloid fibril core formed by necroptosis-mediating RHIM of human RIPK3.
Authors: Xialian Wu / Yeyang Ma / Kun Zhao / Jing Zhang / Yunpeng Sun / Yichen Li / Xingqi Dong / Hong Hu / Jing Liu / Jian Wang / Xia Zhang / Bing Li / Huayi Wang / Dan Li / Bo Sun / Junxia Lu / Cong Liu /
Abstract: Receptor-interacting protein kinases 3 (RIPK3), a central node in necroptosis, polymerizes in response to the upstream signals and then activates its downstream mediator to induce cell death. The ...Receptor-interacting protein kinases 3 (RIPK3), a central node in necroptosis, polymerizes in response to the upstream signals and then activates its downstream mediator to induce cell death. The active polymeric form of RIPK3 has been indicated as the form of amyloid fibrils assembled via its RIP homotypic interaction motif (RHIM). In this study, we combine cryogenic electron microscopy and solid-state NMR to determine the amyloid fibril structure of RIPK3 RHIM-containing C-terminal domain (CTD). The structure reveals a single protofilament composed of the RHIM domain. RHIM forms three β-strands (referred to as strands 1 through 3) folding into an S shape, a distinct fold from that in complex with RIPK1. The consensus tetrapeptide VQVG of RHIM forms strand 2, which zips up strands 1 and 3 via heterozipper-like interfaces. Notably, the RIPK3-CTD fibril, as a physiological fibril, exhibits distinctive assembly compared with pathological fibrils. It has an exceptionally small fibril core and twists in both handedness with the smallest pitch known so far. These traits may contribute to a favorable spatial arrangement of RIPK3 kinase domain for efficient phosphorylation.
History
DepositionOct 16, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 28, 2021Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Receptor-interacting serine/threonine-protein kinase 3
B: Receptor-interacting serine/threonine-protein kinase 3
C: Receptor-interacting serine/threonine-protein kinase 3
D: Receptor-interacting serine/threonine-protein kinase 3
E: Receptor-interacting serine/threonine-protein kinase 3


Theoretical massNumber of molelcules
Total (without water)59,3155
Polymers59,3155
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, dark field beam-tilt electron microscope experiments determined the mass-per-length which indicated a one-fold amyloid structure, X-ray experiment displayed two typical distance ...Evidence: microscopy, dark field beam-tilt electron microscope experiments determined the mass-per-length which indicated a one-fold amyloid structure, X-ray experiment displayed two typical distance of amyloid structure.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9580 Å2
ΔGint-34 kcal/mol
Surface area8520 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)12 / 96structures with the lowest energy
RepresentativeModel #1medoid

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Components

#1: Protein
Receptor-interacting serine/threonine-protein kinase 3 / RIP-like protein kinase 3 / Receptor-interacting protein 3 / RIP-3


Mass: 11863.072 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RIPK3, RIP3 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Rosetta
References: UniProt: Q9Y572, non-specific serine/threonine protein kinase

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Experimental details

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Experiment

ExperimentMethod: SOLID-STATE NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic12D DARR 50ms mixing
1151isotropic12D DARR 200ms mixing
1161isotropic12D DARR 500ms mixing
121isotropic12D NcaCX 50ms mixing
131isotropic12D NCOCX 50ms mixing
141isotropic13D NCACX 50ms mixing
151isotropic13D NCOCX 50ms mixing
1171isotropic1zTEDOR 6.4ms mixing
1191isotropic1zTEDOR 8.5ms mixing
1181isotropic12D RFDR 10.7ms mixing
1201isotropic12D INEPT-TOBSY
162isotropic12D DARR 50ms mixing
172isotropic12D DARR 200ms mixing
182isotropic12D DARR 500ms mixing
1212isotropic1zTEDOR 6.4ms mixing
1222isotropic1zTEDOR 8.5ms mixing
1372isotropic12D NcaCX 50ms mixing
1382isotropic12D NCOCX 50ms mixing
193isotropic12D DARR 50ms mixing
1103isotropic12D DARR 200ms mixing
1113isotropic12D DARR 500ms mixing
1243isotropic1zTEDOR 6.4ms mixing
1263isotropic1zTEDOR 8.5ms mixing
1393isotropic12D NcaCX 50ms mixing
1403isotropic12D NCOCX 50ms mixing
1124isotropic12D DARR 50ms mixing
1134isotropic12D DARR 200ms mixing
1144isotropic12D DARR 500ms mixing
1274isotropic1zTEDOR 6.4ms mixing
1284isotropic1zTEDOR 8.5ms mixing
1294anisotropic12D NHHC
1301isotropic22D DARR 50ms mixing
1311isotropic22D DARR 200ms mixing
1321isotropic22D DARR 500ms mixing
1331isotropic22D NcaCX 50ms mixing
1341isotropic22D NCOCX 50ms mixing
1351isotropic23D NCACX 50ms mixing
1361isotropic23D NCOCX 50ms mixing

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
gel solid15 mg/mL [U-100% 13C; U-100% 15N] human RIPK3 fibrils, 95% H2O/5% D2Ouniformly labeled human RIPK3 with 13C-gulcose and 15N-NH4Cl for carbon and nitrogen source, respectively.uniformly labeled human RIPK3 fibrils95% H2O/5% D2O
gel solid25 mg/mL [U-100% 2-13C Glycerol; U-100% 15N] human RIPK3 fibrils, 95% H2O/5% D2O2-13C glycerol, 15N labeled human RIPK3 with 2-13C glycerol and 15N-NH4Cl for carbon and nitrogen source, respectively.2-13C glycerol, 15N labeled human RIPK3 fibrils95% H2O/5% D2O
gel solid35 mg/mL [U-100% 1, 3-13C Glycerol; U-100% 15N] human RIPK3 fibrils, 95% H2O/5% D2O1, 3-13C glycerol, 15N labeled human RIPK3 with 1, 3-13C glycerol and 15N-NH4Cl for carbon and nitrogen source, respectively.1, 3-13C glycerol, 15N labeled human RIPK3 fibrils95% H2O/5% D2O
gel solid45 mg/mL [U-50% 13C; natural abundance N][natural abandance 13C; U-50% 15N] human RIPK3 fibrils, 95% H2O/5% D2O[U-50% 13C; natural abundance N] labeled human RIPK3 mixed with [natural abandance 13C; U-50% 15N] at 1:1 molar ratio.[U-50% 13C; natural abundance N][natural abandance 13C; U-50% 15N] mixed-labeled human RIPK3 fibrils95% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
5 mg/mLhuman RIPK3 fibrils[U-100% 13C; U-100% 15N]1
5 mg/mLhuman RIPK3 fibrils[U-100% 2-13C Glycerol; U-100% 15N]2
5 mg/mLhuman RIPK3 fibrils[U-100% 1, 3-13C Glycerol; U-100% 15N]3
5 mg/mLhuman RIPK3 fibrils[U-50% 13C; natural abundance N][natural abandance 13C; U-50% 15N]4
Sample conditionsDetails: dialyzed in H2O for 3 days with pH adjusted to 7.5 / Ionic strength: 0 mM / Ionic strength err: 0.5 / Label: H2O / pH: 7.5 / PH err: 0.05 / Pressure: 1 atm / Pressure err: 0.01 / Temperature: 298 K / Temperature err: 0.5

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Bruker AVANCE NEOBrukerAVANCE NEO70013.2 mm MAS E-free H/C/N
Agilent Direct DriveAgilentDirect Drive7002solid state NMR probe

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Processing

NMR software
NameDeveloperClassification
CNSBrunger A. T. et.al.refinement
X-PLOR NIHSchwieters, Kuszewski, Tjandra and Clorestructure calculation
SparkyGoddardchemical shift assignment
SparkyGoddardpeak picking
RefinementMethod: simulated annealing / Software ordinal: 2
NMR representativeSelection criteria: medoid
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 96 / Conformers submitted total number: 12

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