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

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Basic information

Entry
Database: PDB / ID: 5jcf
TitleCrystal structure of chicken MDA5 with 5'p 10-mer dsRNA and ADP-Mg2+ at 2.6 A resolution (orthorhombic form).
Components
  • Melanoma differentiation associated protein-5
  • RNA (5'-R(P*AP*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
  • 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 / RNA helicase activity / RNA helicase / hydrolase activity / innate immune response / DNA binding ...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 / RNA helicase activity / RNA helicase / hydrolase activity / innate immune response / DNA binding / RNA binding / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
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 ...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 / 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 nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / RNA / RNA (> 10) / RNA helicase
Similarity search - Component
Biological speciesGallus gallus (chicken)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 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 1.1Feb 20, 2019Group: Author supporting evidence / Data collection / Source and taxonomy
Category: pdbx_audit_support / pdbx_entity_src_syn
Item: _pdbx_audit_support.funding_organization / _pdbx_entity_src_syn.ncbi_taxonomy_id / _pdbx_entity_src_syn.organism_scientific
Revision 1.2Jan 10, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / 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 / _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*AP*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
B: Melanoma differentiation associated protein-5
C: RNA (5'-R(P*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
D: RNA (5'-R(P*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)177,40512
Polymers176,3716
Non-polymers1,0346
Water3,027168
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*AP*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)88,8676
Polymers88,3503
Non-polymers5173
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5170 Å2
ΔGint-62 kcal/mol
Surface area34510 Å2
MethodPISA
2
B: Melanoma differentiation associated protein-5
C: RNA (5'-R(P*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')
D: 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
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5140 Å2
ΔGint-63 kcal/mol
Surface area34310 Å2
MethodPISA
Unit cell
Length a, b, c (Å)101.920, 132.470, 139.040
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:

Component-ID: 0 / Ens-ID: 1 / Beg auth comp-ID: GLY / Beg label comp-ID: GLY / End auth comp-ID: ASP / End label comp-ID: ASP / Refine code: 0 / Auth seq-ID: 297 - 988 / Label seq-ID: 4 - 695

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

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Components

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Protein , 1 types, 2 molecules AB

#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 / Plasmid: PETM11 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta 2 / References: UniProt: D9N195

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RNA chain , 2 types, 4 molecules XCDY

#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.: 3 / Source method: obtained synthetically
Details: RNA with 5' monophosphate made with T7 polymerase in vitro
Source: (synth.) synthetic construct (others)
#3: RNA chain RNA (5'-R(P*AP*GP*GP*UP*AP*CP*GP*UP*AP*CP*C)-3')


Mass: 3506.154 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: RNA with 5' monophosphate made with T7 polymerase in vitro
Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 174 molecules

#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine 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
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: Zn
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 168 / Source method: isolated from a natural source / Formula: H2O

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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: 2.66 Å3/Da / Density % sol: 53.78 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7
Details: Directly after size exclusion chromatography chMDA5 delta CARD-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 ...Details: Directly after size exclusion chromatography chMDA5 delta CARD-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 (adenosine 5 prime -(beta,gamma-imido)triphosphate lithium salt hydrate ) 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: 2.6→50 Å / Num. obs: 57525 / % possible obs: 98.2 % / Redundancy: 5.06 % / Rmerge(I) obs: 0.096 / Net I/σ(I): 10.3
Reflection shellResolution: 2.6→2.69 Å / Redundancy: 5.21 % / Rmerge(I) obs: 0.992 / Mean I/σ(I) obs: 1.87 / % possible all: 99.2

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Processing

Software
NameVersionClassification
REFMAC5.8.0135refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4GL2

4gl2


Resolution: 2.6→50 Å / Cor.coef. Fo:Fc: 0.927 / Cor.coef. Fo:Fc free: 0.888 / SU B: 37.55 / SU ML: 0.37 / Cross valid method: THROUGHOUT / ESU R: 0.949 / ESU R Free: 0.387 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.31231 2901 5 %RANDOM
Rwork0.27663 ---
obs0.2784 54624 98.22 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 65.634 Å2
Baniso -1Baniso -2Baniso -3
1--1.24 Å20 Å20 Å2
2--5.99 Å2-0 Å2
3----4.75 Å2
Refinement stepCycle: LAST / Resolution: 2.6→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10771 873 58 168 11870
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.01911979
X-RAY DIFFRACTIONr_bond_other_d0.0020.0211239
X-RAY DIFFRACTIONr_angle_refined_deg1.0551.90616284
X-RAY DIFFRACTIONr_angle_other_deg0.9013.00125925
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.58251306
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.86124.453548
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.562152187
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.631586
X-RAY DIFFRACTIONr_chiral_restr0.060.21780
X-RAY DIFFRACTIONr_gen_planes_refined0.0030.0212677
X-RAY DIFFRACTIONr_gen_planes_other0.0010.022706
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_it0.3223.5865266
X-RAY DIFFRACTIONr_mcbond_other0.3223.5865265
X-RAY DIFFRACTIONr_mcangle_it0.5925.3756558
X-RAY DIFFRACTIONr_mcangle_other0.5925.3756559
X-RAY DIFFRACTIONr_scbond_it0.2593.5956713
X-RAY DIFFRACTIONr_scbond_other0.2593.5956712
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other0.4775.3769726
X-RAY DIFFRACTIONr_long_range_B_refined2.27728.20113111
X-RAY DIFFRACTIONr_long_range_B_other2.2328.16313083
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Ens-ID: 1 / Number: 166934 / Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Rms dev position: 0.04 Å / Weight position: 0.05

Dom-IDAuth asym-ID
1A
2B
LS refinement shellResolution: 2.6→2.668 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.418 203 -
Rwork0.401 4035 -
obs--99.23 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.3320.6606-0.80123.43340.47285.5876-0.10750.3885-0.0131-0.3742-0.0211-0.0347-0.0449-0.0360.12860.1781-0.09360.01360.1401-0.01040.0643198.515720.8023137.2338
22.61241.20381.25861.11270.89994.9877-0.15210.0943-0.1014-0.06990.0274-0.03460.4628-0.66230.12470.1939-0.10140.13290.1272-0.02020.1521192.441512.7854153.7573
31.45360.30240.37632.43611.68863.6981-0.10530.0884-0.17320.14190.1008-0.31660.09840.54540.00450.193-0.0464-0.01470.21750.07560.1135219.683926.0073176.5978
45.5951-0.5270.82850.8183-0.32242.90260.03980.4349-0.0515-0.1612-0.0721-0.04330.2181-0.03050.03230.2515-0.0360.10890.0495-0.0340.1011199.018414.2423158.299
57.74871.8855-0.37572.61820.1966.94950.21681.06870.7244-0.2426-0.11340.158-0.7315-0.4015-0.10340.66490.001-0.01480.25270.1560.3587215.055651.8765165.2358
61.21030.06962.79133.6165-0.5726.6173-0.2344-0.14670.1640.3154-0.01730.3083-0.6625-0.30310.25170.2104-0.07020.10760.125-0.05950.1839202.147530.4445163.4543
72.3785-0.5941-0.90682.96920.04045.7426-0.0584-0.34360.04410.2721-0.0544-0.0226-0.105-0.05170.11290.15020.0608-0.00840.11750.01950.0673183.119820.7177210.4073
83.0541-0.92351.24951.1565-0.7964.0917-0.0914-0.0424-0.08630.0651-0.02610.00050.45490.68450.11760.20650.0810.13520.14430.00970.1199189.043212.4608193.5437
91.4491-0.41490.5782.7396-1.71493.5361-0.0977-0.0619-0.2011-0.15540.11030.33710.0598-0.685-0.01270.18020.0458-0.02990.3003-0.07570.1099161.945825.7212170.9999
104.97480.57730.86390.74280.33093.03470.0052-0.4719-0.07260.1517-0.05170.030.1733-0.06010.04650.2310.02570.09630.0510.03490.1346182.700714.103189.2867
118.9018-1.57910.25772.64340.51834.84520.221-1.09250.6220.3113-0.1802-0.0388-0.5940.0459-0.04090.62440.01040.00110.3144-0.09860.3488166.339951.6624182.4215
120.96350.16552.43544.73780.24527.0793-0.17450.12820.232-0.3059-0.0302-0.1853-0.65040.00540.20470.10820.06250.07450.18280.1370.2418178.783130.1718184.4469
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 458
2X-RAY DIFFRACTION2A459 - 541
3X-RAY DIFFRACTION3A542 - 746
4X-RAY DIFFRACTION4A747 - 866
5X-RAY DIFFRACTION5A867 - 1100
6X-RAY DIFFRACTION6X1 - 10
7X-RAY DIFFRACTION6Y0 - 10
8X-RAY DIFFRACTION7B1 - 458
9X-RAY DIFFRACTION8B459 - 541
10X-RAY DIFFRACTION9B542 - 746
11X-RAY DIFFRACTION10B747 - 866
12X-RAY DIFFRACTION11B867 - 1100
13X-RAY DIFFRACTION12C1 - 10
14X-RAY DIFFRACTION12D1 - 10

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