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- PDB-3h2l: Crystal structure of HCV NS5B polymerase in complex with a novel ... -

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Basic information

Entry
Database: PDB / ID: 3h2l
TitleCrystal structure of HCV NS5B polymerase in complex with a novel bicyclic dihydro-pyridinone inhibitor
ComponentsNS5B polymerase
KeywordsTRANSFERASE / PROTEIN-LIGAND COMPLEX / HEPATITIS C / RNA REPLICATION / RNA-BINDING / RNA-DIRECTED RNA POLYMERASE / Acetylation / Apoptosis / ATP-binding
Function / homology
Function and homology information


hepacivirin / host cell mitochondrial membrane / host cell lipid droplet / symbiont-mediated suppression of host TRAF-mediated signal transduction / transformation of host cell by virus / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / SH3 domain binding / nucleoside-triphosphate phosphatase ...hepacivirin / host cell mitochondrial membrane / host cell lipid droplet / symbiont-mediated suppression of host TRAF-mediated signal transduction / transformation of host cell by virus / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / SH3 domain binding / nucleoside-triphosphate phosphatase / channel activity / monoatomic ion transmembrane transport / viral nucleocapsid / clathrin-dependent endocytosis of virus by host cell / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / RNA helicase activity / host cell perinuclear region of cytoplasm / host cell endoplasmic reticulum membrane / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / induction by virus of host autophagy / ribonucleoprotein complex / RNA-directed RNA polymerase / viral RNA genome replication / cysteine-type endopeptidase activity / serine-type endopeptidase activity / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / fusion of virus membrane with host endosome membrane / viral envelope / host cell nucleus / virion attachment to host cell / apoptotic process / host cell plasma membrane / structural molecule activity / virion membrane / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane
Similarity search - Function
Hepatitus C virus, Non-structural 5a protein, C-terminal / Hepatitis C virus NS5A, 1B domain superfamily / Hepatitis C virus non-structural protein NS2, N-terminal domain / Hepatitis C virus non-structural protein NS2 / HCV NS5a protein C-terminal region / Hepatitis C virus, Non-structural protein NS4b / Hepatitis C virus, Core protein, N-terminal / Hepatitis C virus non-structural protein NS2, C-terminal domain / Hepatitis C virus core protein, chain A superfamily / : ...Hepatitus C virus, Non-structural 5a protein, C-terminal / Hepatitis C virus NS5A, 1B domain superfamily / Hepatitis C virus non-structural protein NS2, N-terminal domain / Hepatitis C virus non-structural protein NS2 / HCV NS5a protein C-terminal region / Hepatitis C virus, Non-structural protein NS4b / Hepatitis C virus, Core protein, N-terminal / Hepatitis C virus non-structural protein NS2, C-terminal domain / Hepatitis C virus core protein, chain A superfamily / : / Hepatitis C virus non-structural protein NS4b / Hepatitis C virus capsid protein / Hepatitis C virus, Non-structural 5a protein / Hepatitis C virus, Non-structural 5a protein, domain 1a / Hepatitis C virus non-structural 5a, 1B domain / NS5A domain 1a superfamily / Hepatitis C virus non-structural 5a zinc finger domain / Hepatitis C virus non-structural 5a domain 1b / Hepatitis C virus, Non-structural protein NS2 / : / NS3 RNA helicase, C-terminal helical domain / Hepacivirus nonstructural protein 2 (NS2) protease domain profile. / Hepatitis C virus non-structural 5a protein membrane anchor / Hepatitis C virus, Non-structural protein NS4a / Hepatitis C virus non-structural protein NS4a / Hepatitis C virus, Core protein, C-terminal / Hepatitis C virus core protein / Hepatitis C virus, Non-structural protein E2/NS1 / Hepatitis C virus non-structural protein E2/NS1 / Hepatitis C virus, Envelope glycoprotein E1 / Hepatitis C virus envelope glycoprotein E1 / RNA dependent RNA polymerase, hepatitis C virus / Viral RNA dependent RNA polymerase / Hepatitis C virus, NS3 protease, Peptidase S29 / Hepacivirus/Pegivirus NS3 protease domain profile. / Hepatitis C virus NS3 protease / Reverse transcriptase/Diguanylate cyclase domain / DEAD box, Flavivirus / Flavivirus DEAD 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 / Reverse transcriptase/Diguanylate cyclase domain / Alpha-Beta Plaits / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-YAK / Genome polyprotein
Similarity search - Component
Biological speciesHepatitis C virus
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.9 Å
AuthorsHan, Q. / Showalter, R.E. / Zhou, Q. / Kissinger, C.R.
CitationJournal: Bioorg.Med.Chem.Lett. / Year: 2009
Title: Discovery of tricyclic 5,6-dihydro-1H-pyridin-2-ones as novel, potent, and orally bioavailable inhibitors of HCV NS5B polymerase.
Authors: Ruebsam, F. / Murphy, D.E. / Tran, C.V. / Li, L.S. / Zhao, J. / Dragovich, P.S. / McGuire, H.M. / Xiang, A.X. / Sun, Z. / Ayida, B.K. / Blazel, J.K. / Kim, S.H. / Zhou, Y. / Han, Q. / ...Authors: Ruebsam, F. / Murphy, D.E. / Tran, C.V. / Li, L.S. / Zhao, J. / Dragovich, P.S. / McGuire, H.M. / Xiang, A.X. / Sun, Z. / Ayida, B.K. / Blazel, J.K. / Kim, S.H. / Zhou, Y. / Han, Q. / Kissinger, C.R. / Webber, S.E. / Showalter, R.E. / Shah, A.M. / Tsan, M. / Patel, R.A. / Thompson, P.A. / Lebrun, L.A. / Hou, H.J. / Kamran, R. / Sergeeva, M.V. / Bartkowski, D.M. / Nolan, T.G. / Norris, D.A. / Khandurina, J. / Brooks, J. / Okamoto, E. / Kirkovsky, L.
History
DepositionApr 14, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 8, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 13, 2021Group: Database references / Derived calculations / Category: database_2 / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.3Feb 21, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Revision 1.4Apr 3, 2024Group: Refinement description / Category: pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: NS5B polymerase
B: NS5B polymerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)129,7614
Polymers128,6912
Non-polymers1,0692
Water8,737485
1
A: NS5B polymerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,8802
Polymers64,3461
Non-polymers5351
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: NS5B polymerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,8802
Polymers64,3461
Non-polymers5351
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)86.548, 106.908, 126.575
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein NS5B polymerase


Mass: 64345.738 Da / Num. of mol.: 2 / Fragment: Catalytic domain: UNP residues 2420-2989 / Mutation: R544Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hepatitis C virus (isolate BK) / Strain: genotype 1b, isolate BK / Plasmid: pET21a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)STAR / References: UniProt: P26663, RNA-directed RNA polymerase
#2: Chemical ChemComp-YAK / N-{3-[(4aR,7aS)-1-(4-fluorobenzyl)-4-hydroxy-2-oxo-2,4a,5,6,7,7a-hexahydro-1H-cyclopenta[b]pyridin-3-yl]-1,1-dioxido-2H-1,2,4-benzothiadiazin-7-yl}methanesulfonamide


Mass: 534.580 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C23H23FN4O6S2
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 485 / 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: 2.28 Å3/Da / Density % sol: 45.94 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 4.7
Details: 20% PEG 4000, 50 mM Ammonium sulfate, 100 mM Sodium acetate pH 4.7, 5 mM DTT, transferred to pH 7.6 for soaking, VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RUH3R / Wavelength: 1.5418 Å
DetectorType: RIGAKU RAXIS IV / Detector: IMAGE PLATE / Date: Feb 10, 2007 / Details: Mirrors
RadiationMonochromator: Osmic mirrors / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.9→45.5 Å / Num. all: 88942 / Num. obs: 88942 / % possible obs: 95.9 % / Observed criterion σ(F): 0 / Net I/σ(I): 14.9
Reflection shellResolution: 1.9→1.95 Å / Mean I/σ(I) obs: 1.2 / Num. unique all: 6160 / % possible all: 91

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Processing

Software
NameVersionClassification
HKL-2000data collection
EPMRphasing
REFMAC5.2.0004refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: HCV NS5B POLYMERASE

Resolution: 1.9→45.5 Å / Cor.coef. Fo:Fc: 0.951 / Cor.coef. Fo:Fc free: 0.933 / SU B: 5.456 / SU ML: 0.152 / Isotropic thermal model: ISOTROPIC / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.191 / ESU R Free: 0.168 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.25825 4427 5 %RANDOM
Rwork0.2206 ---
all0.22249 88942 --
obs0.22249 88942 95.9 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 37.064 Å2
Baniso -1Baniso -2Baniso -3
1--1.5 Å20 Å20 Å2
2--3.39 Å20 Å2
3----1.9 Å2
Refinement stepCycle: LAST / Resolution: 1.9→45.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8689 0 72 485 9246
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0228972
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.1631.97412205
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.38651128
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.3422.873362
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.874151523
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.8141570
X-RAY DIFFRACTIONr_chiral_restr0.0740.21381
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.026722
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined0.1940.24293
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined0.2960.26202
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1320.2638
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.1860.250
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1280.214
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.4543.55750
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it2.05949032
X-RAY DIFFRACTIONr_scbond_it2.0864.53723
X-RAY DIFFRACTIONr_scangle_it2.87253164
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.9→1.95 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.4 314 -
Rwork0.362 5846 -
obs--90.98 %

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