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- PDB-5h8o: Crystal structure of an ASC-binding nanobody in complex with the ... -

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Basic information

Entry
Database: PDB / ID: 5h8o
TitleCrystal structure of an ASC-binding nanobody in complex with the CARD domain of ASC
Components
  • Apoptosis-associated speck-like protein containing a CARD
  • VHH nanobody
KeywordsIMMUNE SYSTEM / VHH nanobody / ASC-binding / antibody fragment / inflammasome
Function / homology
Function and homology information


Pyrin domain binding / NLRP6 inflammasome complex / myosin I binding / positive regulation of antigen processing and presentation of peptide antigen via MHC class II / myeloid dendritic cell activation involved in immune response / regulation of intrinsic apoptotic signaling pathway / myeloid dendritic cell activation / IkappaB kinase complex / The AIM2 inflammasome / AIM2 inflammasome complex ...Pyrin domain binding / NLRP6 inflammasome complex / myosin I binding / positive regulation of antigen processing and presentation of peptide antigen via MHC class II / myeloid dendritic cell activation involved in immune response / regulation of intrinsic apoptotic signaling pathway / myeloid dendritic cell activation / IkappaB kinase complex / The AIM2 inflammasome / AIM2 inflammasome complex / macropinocytosis / icosanoid biosynthetic process / interleukin-6 receptor binding / NLRP1 inflammasome complex / canonical inflammasome complex / NLRP3 inflammasome complex assembly / positive regulation of adaptive immune response / NLRP3 inflammasome complex / BMP receptor binding / osmosensory signaling pathway / negative regulation of protein serine/threonine kinase activity / negative regulation of interferon-beta production / CLEC7A/inflammasome pathway / positive regulation of cysteine-type endopeptidase activity / regulation of tumor necrosis factor-mediated signaling pathway / positive regulation of extrinsic apoptotic signaling pathway / positive regulation of macrophage cytokine production / pattern recognition receptor signaling pathway / negative regulation of NF-kappaB transcription factor activity / positive regulation of actin filament polymerization / tropomyosin binding / positive regulation of activated T cell proliferation / pyroptotic inflammatory response / positive regulation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of release of cytochrome c from mitochondria / cysteine-type endopeptidase activator activity involved in apoptotic process / positive regulation of interleukin-10 production / The NLRP3 inflammasome / intrinsic apoptotic signaling pathway by p53 class mediator / cellular response to interleukin-1 / negative regulation of cytokine production involved in inflammatory response / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / positive regulation of T cell migration / Purinergic signaling in leishmaniasis infection / positive regulation of chemokine production / negative regulation of canonical NF-kappaB signal transduction / positive regulation of phagocytosis / positive regulation of defense response to virus by host / tumor necrosis factor-mediated signaling pathway / activation of innate immune response / positive regulation of interleukin-1 beta production / regulation of autophagy / positive regulation of interleukin-8 production / positive regulation of JNK cascade / regulation of protein stability / protein homooligomerization / positive regulation of DNA-binding transcription factor activity / positive regulation of inflammatory response / positive regulation of non-canonical NF-kappaB signal transduction / positive regulation of interleukin-6 production / positive regulation of type II interferon production / activation of cysteine-type endopeptidase activity involved in apoptotic process / SARS-CoV-1 activates/modulates innate immune responses / azurophil granule lumen / positive regulation of tumor necrosis factor production / positive regulation of T cell activation / cellular response to tumor necrosis factor / positive regulation of NF-kappaB transcription factor activity / protease binding / cellular response to lipopolysaccharide / secretory granule lumen / positive regulation of canonical NF-kappaB signal transduction / defense response to virus / defense response to Gram-negative bacterium / transmembrane transporter binding / microtubule / positive regulation of ERK1 and ERK2 cascade / protein dimerization activity / defense response to Gram-positive bacterium / positive regulation of apoptotic process / Golgi membrane / innate immune response / neuronal cell body / Neutrophil degranulation / nucleolus / apoptotic process / enzyme binding / signal transduction / endoplasmic reticulum / protein homodimerization activity / protein-containing complex / mitochondrion / extracellular region / nucleoplasm / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
CARD8/ASC/NALP1, CARD domain / : / DAPIN domain / DAPIN domain profile. / PAAD/DAPIN/Pyrin domain / PAAD/DAPIN/Pyrin domain / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily
Similarity search - Domain/homology
Apoptosis-associated speck-like protein containing a CARD
Similarity search - Component
Biological speciesLama glama (llama)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 4.206 Å
AuthorsLu, A. / Schmidt, F.I. / Ruan, J. / Tang, C. / Wu, H. / Ploegh, H.L.
CitationJournal: J.Exp.Med. / Year: 2016
Title: A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.
Authors: Schmidt, F.I. / Lu, A. / Chen, J.W. / Ruan, J. / Tang, C. / Wu, H. / Ploegh, H.L.
History
DepositionDec 23, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 6, 2016Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2016Group: Database references
Revision 1.2May 18, 2016Group: Derived calculations

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: VHH nanobody
B: Apoptosis-associated speck-like protein containing a CARD


Theoretical massNumber of molelcules
Total (without water)21,7172
Polymers21,7172
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)53.625, 30.996, 79.244
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number3
Space group name H-MP121

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Components

#1: Antibody VHH nanobody


Mass: 12253.590 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#2: Protein Apoptosis-associated speck-like protein containing a CARD / hASC / Caspase recruitment domain-containing protein 5 / PYD and CARD domain-containing protein / ...hASC / Caspase recruitment domain-containing protein 5 / PYD and CARD domain-containing protein / Target of methylation-induced silencing 1


Mass: 9463.741 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PYCARD, ASC, CARD5, TMS1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9ULZ3

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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: 3.06 Å3/Da / Density % sol: 59.82 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 3.5
Details: 0.1 M citric acid at pH 3.5 and 3 M sodium chloride

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9791 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Feb 19, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9791 Å / Relative weight: 1
ReflectionResolution: 4.2→79.24 Å / Num. obs: 2025 / % possible obs: 99 % / Redundancy: 6.4 % / Biso Wilson estimate: 60.49 Å2 / CC1/2: 0.736 / Rmerge(I) obs: 0.824 / Rpim(I) all: 0.349 / Net I/σ(I): 2.1 / Num. measured all: 12955
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allCC1/2Rpim(I) all% possible all
4.2-4.436.71.158219672940.6380.477100
13.28-79.245.90.3133.2463780.9130.13999.2

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
Aimless0.1.27data scaling
PHASERphasing
PHENIX1.9_1692refinement
PDB_EXTRACT3.15data extraction
iMOSFLMdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 4.206→79.24 Å / SU ML: 0.6 / Cross valid method: FREE R-VALUE / σ(F): 0 / Phase error: 36.37 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3556 109 5.38 %
Rwork0.2998 1916 -
obs0.303 2025 99.02 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 207.51 Å2 / Biso mean: 86.866 Å2 / Biso min: 22.67 Å2
Refinement stepCycle: final / Resolution: 4.206→79.24 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1051 0 0 0 1051
Num. residues----181
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0051063
X-RAY DIFFRACTIONf_angle_d1.1511455
X-RAY DIFFRACTIONf_chiral_restr0.038172
X-RAY DIFFRACTIONf_plane_restr0.005204
X-RAY DIFFRACTIONf_dihedral_angle_d12.084285
LS refinement shellHighest resolution: 4.2061 Å
RfactorNum. reflection% reflection
Rfree0.3556 109 -
Rwork0.2998 1916 -
obs--99 %
Refinement TLS params.Method: refined / Origin x: -1.7913 Å / Origin y: 7.7117 Å / Origin z: 22.6124 Å
111213212223313233
T0.3714 Å20.0915 Å2-0.2352 Å2-0.4568 Å2-0.0339 Å2---0.1266 Å2
L2.8005 °20.8979 °20.6353 °2-1.9681 °20.5119 °2--1.1547 °2
S-0.362 Å °0.725 Å °-0.2578 Å °-0.4619 Å °0.296 Å °-0.7249 Å °0.0839 Å °-0.403 Å °-0.0317 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA3 - 113
2X-RAY DIFFRACTION1allB115 - 195

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