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- PDB-5jch: Crystal structure of chicken MDA5 with 5'p 10-mer dsRNA and ADP-M... -

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Basic information

Entry
Database: PDB / ID: 5jch
TitleCrystal structure of chicken MDA5 with 5'p 10-mer dsRNA and ADP-Mg2+ at 2.95 A resolution (untwinned).
Components
  • Melanoma differentiation associated protein-5
  • RNA (5'-R(P*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
KeywordsIMMUNE SYSTEM / Innate immune pattern recognition receptor / RIG-I like helicase / dsRNA dependent ATPase / zinc-containing CTD domain
Function / homology
Function and homology information


Caspase-8 and -10 mediated induction of NF-kB / RLR (RIG-like receptor) mediated induction of IFN alpha/beta / TRAF mediated activation of IRF / Negative Regulation of MDA5 signaling / TRAF6 mediated NF-kB activation / defense response to virus / RNA helicase activity / hydrolase activity / RNA helicase / innate immune response ...Caspase-8 and -10 mediated induction of NF-kB / RLR (RIG-like receptor) mediated induction of IFN alpha/beta / TRAF mediated activation of IRF / Negative Regulation of MDA5 signaling / TRAF6 mediated NF-kB activation / defense response to virus / RNA helicase activity / hydrolase activity / RNA helicase / innate immune response / DNA binding / RNA binding / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
phosphoenolpyruvate carboxylase, domain 3 - #30 / phosphoenolpyruvate carboxylase, domain 3 / RIG-I-like receptor, C-terminal regulatory domain / Metal Binding Protein, Guanine Nucleotide Exchange Factor; Chain A / RIG-I-like receptor, C-terminal / RIG-I receptor C-terminal domain / RIG-I-like receptor, C-terminal regulatory domain / RIG-I-like receptor, C-terminal domain superfamily / : / C-terminal domain of RIG-I ...phosphoenolpyruvate carboxylase, domain 3 - #30 / phosphoenolpyruvate carboxylase, domain 3 / RIG-I-like receptor, C-terminal regulatory domain / Metal Binding Protein, Guanine Nucleotide Exchange Factor; Chain A / RIG-I-like receptor, C-terminal / RIG-I receptor C-terminal domain / RIG-I-like receptor, C-terminal regulatory domain / RIG-I-like receptor, C-terminal domain superfamily / : / C-terminal domain of RIG-I / RIG-I-like receptor (RLR) C-terminal regulatory (CTR) domain profile. / Caspase recruitment domain / Caspase recruitment domain / Helicase/UvrB, N-terminal / Type III restriction enzyme, res subunit / Death-like domain superfamily / Beta Complex / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Armadillo-type fold / P-loop containing nucleotide triphosphate hydrolases / Up-down Bundle / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / RNA / RNA helicase
Similarity search - Component
Biological speciesGallus gallus (chicken)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.95 Å
AuthorsCusack, S. / Uchikawa, E.
Funding support France, 1items
OrganizationGrant numberCountry
European Research Council322586 France
CitationJournal: Mol.Cell / Year: 2016
Title: Structural Analysis of dsRNA Binding to Anti-viral Pattern Recognition Receptors LGP2 and MDA5.
Authors: Uchikawa, E. / Lethier, M. / Malet, H. / Brunel, J. / Gerlier, D. / Cusack, S.
History
DepositionApr 15, 2016Deposition site: RCSB / Processing site: PDBE
Revision 1.0Jun 1, 2016Provider: repository / Type: Initial release
Revision 1.1Jan 10, 2024Group: Author supporting evidence / Data collection ...Author supporting evidence / Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_audit_support / pdbx_initial_refinement_model / struct_conn / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Melanoma differentiation associated protein-5
X: RNA (5'-R(P*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
Y: RNA (5'-R(P*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
B: Melanoma differentiation associated protein-5
U: RNA (5'-R(P*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
V: RNA (5'-R(P*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)177,07612
Polymers176,0426
Non-polymers1,0346
Water00
1
A: Melanoma differentiation associated protein-5
X: RNA (5'-R(P*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
Y: RNA (5'-R(P*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)88,5386
Polymers88,0213
Non-polymers5173
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5170 Å2
ΔGint-62 kcal/mol
Surface area34200 Å2
MethodPISA
2
B: Melanoma differentiation associated protein-5
U: RNA (5'-R(P*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
V: RNA (5'-R(P*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)88,5386
Polymers88,0213
Non-polymers5173
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5190 Å2
ΔGint-63 kcal/mol
Surface area34040 Å2
MethodPISA
Unit cell
Length a, b, c (Å)72.080, 139.730, 103.190
Angle α, β, γ (deg.)90.00, 110.14, 90.00
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:

Component-ID: _ / Ens-ID: 1 / Beg auth comp-ID: ASP / Beg label comp-ID: ASP / End auth comp-ID: ASP / End label comp-ID: ASP / Refine code: _ / Auth seq-ID: 298 - 988 / Label seq-ID: 5 - 695

Dom-IDAuth asym-IDLabel asym-ID
1AA
2BD

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Components

#1: Protein Melanoma differentiation associated protein-5


Mass: 81667.008 Da / Num. of mol.: 2
Mutation: N-terminal deletion of 1-297; GAMG from tag at N-terminus; C-terminal deletion of 995-1001; Point mutation E436Q.
Source method: isolated from a genetically manipulated source
Details: Chicken MDA5 helicase and CTD domains / Source: (gene. exp.) Gallus gallus (chicken) / Gene: MDA5 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta 2 / References: UniProt: D9N195
#2: RNA chain
RNA (5'-R(P*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')


Mass: 3176.948 Da / Num. of mol.: 4 / Source method: obtained synthetically
Details: Synthetic dsRNA obtained by in vitro T7 transcription
Source: (synth.) synthetic construct (others)
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.77 Å3/Da / Density % sol: 55.62 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7
Details: Directly after size exclusion chromatography chMDA5 deltaCARD-Q was mixed with 10 bp dsRNA in a 1:1 molar ratio and incubated for 30 minutes on ice. The complex was concentrated to around 10 ...Details: Directly after size exclusion chromatography chMDA5 deltaCARD-Q was mixed with 10 bp dsRNA in a 1:1 molar ratio and incubated for 30 minutes on ice. The complex was concentrated to around 10 mg/ml and 2 mM AMPPNP was added. Sample and reservoir buffer (0.025 M Bis-Tris pH 6.5, 0.075 M succinic acid pH 7.0, 12-14% PEG 3350, 2% sucrose) were mixed in a 2:1 ratio. Three hours after setup, cover glasses with drops were transferred from a reservoir containing 12-14% PEG 3350 to one containing 8% PEG 3350. Crystals grew in one week at 20 C and were harvested in cryo-protectant solution (0.025 M Bis-Tris pH 6.5, 0.075 M succinic acid pH 7.0, 25% 3350, 10% ethylene glycol) before flash freezing with liquid nitrogen.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 0.9763 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Feb 10, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 2.95→50 Å / Num. obs: 38060 / % possible obs: 94 % / Redundancy: 5.2 % / Rmerge(I) obs: 0.094 / Net I/σ(I): 9.94
Reflection shellResolution: 2.95→3.06 Å / Redundancy: 5.05 % / Rmerge(I) obs: 0.873 / Mean I/σ(I) obs: 1.78 / % possible all: 96.6

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Processing

Software
NameVersionClassification
REFMAC5.8.0131refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5JC3

5jc3


Resolution: 2.95→50 Å / Cor.coef. Fo:Fc: 0.917 / Cor.coef. Fo:Fc free: 0.908 / SU B: 30.813 / SU ML: 0.491 / Cross valid method: THROUGHOUT / ESU R Free: 0.47 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.27046 1907 5 %RANDOM
Rwork0.25582 ---
obs0.25657 36153 94.16 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 81.287 Å2
Baniso -1Baniso -2Baniso -3
1-6.11 Å20 Å2-3.92 Å2
2---7.47 Å20 Å2
3---3.33 Å2
Refinement stepCycle: 1 / Resolution: 2.95→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10749 854 58 0 11661
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.01911930
X-RAY DIFFRACTIONr_bond_other_d0.0010.0211228
X-RAY DIFFRACTIONr_angle_refined_deg1.0141.90816210
X-RAY DIFFRACTIONr_angle_other_deg0.8763.00125900
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.3151302
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.80624.401543
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.722152186
X-RAY DIFFRACTIONr_dihedral_angle_4_deg13.8721586
X-RAY DIFFRACTIONr_chiral_restr0.0570.21773
X-RAY DIFFRACTIONr_gen_planes_refined0.0030.0212617
X-RAY DIFFRACTIONr_gen_planes_other0.0010.022695
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.4358.2465250
X-RAY DIFFRACTIONr_mcbond_other1.4338.2455249
X-RAY DIFFRACTIONr_mcangle_it2.62512.3516538
X-RAY DIFFRACTIONr_mcangle_other2.62512.3526539
X-RAY DIFFRACTIONr_scbond_it1.0198.0716680
X-RAY DIFFRACTIONr_scbond_other1.0198.0716679
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other1.91312.0089672
X-RAY DIFFRACTIONr_long_range_B_refined4.01362.09312905
X-RAY DIFFRACTIONr_long_range_B_other4.01362.09412906
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Ens-ID: 1 / Number: 167114 / Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Rms dev position: 0.01 Å / Weight position: 0.05

Dom-IDAuth asym-ID
1A
2B
LS refinement shellResolution: 2.95→3.027 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.421 154 -
Rwork0.423 2716 -
obs--96.5 %

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