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- PDB-4obc: Crystal structure of HCV polymerase NS5b genotype 2a JFH-1 isolat... -

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Basic information

Entry
Database: PDB / ID: 4obc
TitleCrystal structure of HCV polymerase NS5b genotype 2a JFH-1 isolate with the S15G, C223H, V321I resistance mutations against the guanosine analog GS-0938 (PSI-3529238)
ComponentsRNA-directed RNA polymeraseRNA-dependent RNA polymerase
KeywordsViral Protein / transferase / Hepatitis / HCV / viral polymerase / RNA-dependent-RNA-polymerase / RdRp / resistance / nucleotide analog inhibitor
Function / homology
Function and homology information


negative regulation of autophagy of mitochondrion / : / hepacivirin / host cell mitochondrial membrane / host cell lipid droplet / symbiont-mediated suppression of host TRAF-mediated signal transduction / transformation of host cell by virus / Dectin-2 family / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 activity ...negative regulation of autophagy of mitochondrion / : / hepacivirin / host cell mitochondrial membrane / host cell lipid droplet / symbiont-mediated suppression of host TRAF-mediated signal transduction / transformation of host cell by virus / Dectin-2 family / 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 / negative regulation of autophagy / SH3 domain binding / : / nucleoside-triphosphate phosphatase / protein complex oligomerization / monoatomic ion channel activity / clathrin-dependent endocytosis of virus by host cell / viral nucleocapsid / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / RNA helicase activity / host cell endoplasmic reticulum membrane / host cell perinuclear region of cytoplasm / RNA helicase / induction by virus of host autophagy / ribonucleoprotein complex / RNA-directed RNA polymerase / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / serine-type endopeptidase activity / fusion of virus membrane with host endosome membrane / viral envelope / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / host cell nucleus / structural molecule activity / virion attachment to host cell / host cell plasma membrane / virion membrane / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane / identical protein binding
Similarity search - Function
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, C-terminal domain / 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 NS4b ...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, C-terminal domain / 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 NS4b / Hepatitis C virus capsid protein / Hepatitis C virus, Non-structural protein NS2 / 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 protein membrane anchor / Hepatitis C virus non-structural 5a zinc finger domain / Hepatitis C virus non-structural 5a domain 1b / Hepacivirus nonstructural protein 2 (NS2) protease domain profile. / 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 / Hepatitis C virus NS3 protease / Hepacivirus/Pegivirus NS3 protease domain profile. / 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 / 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
Similarity search - Domain/homology
Chem-PG6 / Genome polyprotein
Similarity search - Component
Biological speciesHepatitis C virus JFH-1
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.5 Å
AuthorsEdwards, T.E. / Abendroth, J. / Appleby, T.C.
CitationJournal: Antimicrob.Agents Chemother. / Year: 2014
Title: Molecular and Structural Basis for the Roles of Hepatitis C Virus Polymerase NS5B Amino Acids 15, 223, and 321 in Viral Replication and Drug Resistance.
Authors: Lam, A.M. / Edwards, T.E. / Mosley, R.T. / Murakami, E. / Bansal, S. / Lugo, C. / Bao, H. / Otto, M.J. / Sofia, M.J. / Furman, P.A.
History
DepositionJan 7, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 10, 2014Provider: repository / Type: Initial release
Revision 1.1Oct 29, 2014Group: Database references
Revision 1.2Nov 22, 2017Group: Refinement description / Category: software
Revision 1.3Sep 20, 2023Group: 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_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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-directed RNA polymerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,5016
Polymers64,8191
Non-polymers6815
Water3,711206
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)140.208, 140.208, 92.567
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number170
Space group name H-MP65

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Components

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Protein , 1 types, 1 molecules A

#1: Protein RNA-directed RNA polymerase / RNA-dependent RNA polymerase / NS5B / p68


Mass: 64819.379 Da / Num. of mol.: 1 / Mutation: S15G E86Q E87Q C223H V321I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hepatitis C virus JFH-1 / Strain: JFH-1 / Gene: POLG_HCVJF / Plasmid: V5854 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta DE3 / References: UniProt: Q99IB8, RNA-directed RNA polymerase

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Non-polymers , 5 types, 211 molecules

#2: Chemical ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID / MES (buffer)


Mass: 195.237 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H13NO4S / Comment: pH buffer*YM
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-PG6 / 1-(2-METHOXY-ETHOXY)-2-{2-[2-(2-METHOXY-ETHOXY]-ETHOXY}-ETHANE / Polyethylene glycol


Mass: 266.331 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C12H26O6
#5: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 206 / 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: 4.05 Å3/Da / Density % sol: 69.64 %
Crystal growTemperature: 289 K / Method: vapor diffusion, sitting drop / pH: 8
Details: NS5b VCID 5854 at 4.25 mg/mL in 5 mM Tris pH 7.5, 200 mM NH4OAc, 1 mM EDTA 1 mM DTT against PACT screen condition D5, 25% PEG 1500, 0.1M MMT Malic Acid, MES, Tris Buffer pH 8.0 supplemented ...Details: NS5b VCID 5854 at 4.25 mg/mL in 5 mM Tris pH 7.5, 200 mM NH4OAc, 1 mM EDTA 1 mM DTT against PACT screen condition D5, 25% PEG 1500, 0.1M MMT Malic Acid, MES, Tris Buffer pH 8.0 supplemented with 20% glycerol as cryo-protectant, crystal tracking ID 223572d5, puck ID cps0237-1, VAPOR DIFFUSION, SITTING DROP, temperature 289K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-2 / Wavelength: 0.97946 Å
DetectorType: MARMOSAIC 325 mm CCD / Detector: CCD / Date: Jun 30, 2011
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97946 Å / Relative weight: 1
ReflectionResolution: 2.5→50 Å / Num. obs: 35841 / % possible obs: 99.7 % / Redundancy: 7.8 % / Rmerge(I) obs: 0.085 / Χ2: 0.99 / Net I/σ(I): 21.9
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique allΧ2% possible all
2.5-2.573.70.6382.328770.95597.3
2.57-2.664.50.52129710.95799.3
2.66-2.755.60.43129930.991100
2.75-2.867.50.32429580.966100
2.86-2.9990.25130120.986100
2.99-3.1590.18129561.009100
3.15-3.3590.12829870.962100
3.35-3.6190.08829950.958100
3.61-3.9790.06929810.977100
3.97-4.5490.05530301.045100
4.54-5.7290.05330001.026100
5.72-508.80.04630811.00399.7

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Phasing

PhasingMethod: molecular replacement
Phasing MRRfactor: 50.11 / Model details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation3 Å41.12 Å
Translation3 Å41.12 Å

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
PHASER2.1.4phasing
REFMAC5.5.0109refinement
PDB_EXTRACT3.14data extraction
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1YUY

1yuy


Resolution: 2.5→50 Å / Cor.coef. Fo:Fc: 0.948 / Cor.coef. Fo:Fc free: 0.931 / WRfactor Rfree: 0.209 / WRfactor Rwork: 0.1769 / Occupancy max: 1 / Occupancy min: 0.5 / FOM work R set: 0.8503 / SU B: 6.803 / SU ML: 0.151 / SU R Cruickshank DPI: 0.2565 / SU Rfree: 0.2079 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.257 / ESU R Free: 0.208 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2234 1792 5 %RANDOM
Rwork0.1882 ---
obs0.1899 35745 99.58 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso max: 86.42 Å2 / Biso mean: 39.3253 Å2 / Biso min: 18.41 Å2
Baniso -1Baniso -2Baniso -3
1-1.88 Å20.94 Å20 Å2
2--1.88 Å20 Å2
3----2.81 Å2
Refinement stepCycle: LAST / Resolution: 2.5→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4277 0 43 206 4526
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0130.0224426
X-RAY DIFFRACTIONr_angle_refined_deg1.4211.9726017
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.8485555
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.8522.216176
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.99815713
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.8591539
X-RAY DIFFRACTIONr_chiral_restr0.0870.2682
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.0213311
X-RAY DIFFRACTIONr_mcbond_it0.7411.52776
X-RAY DIFFRACTIONr_mcangle_it1.45224482
X-RAY DIFFRACTIONr_scbond_it2.131650
X-RAY DIFFRACTIONr_scangle_it3.5854.51534
LS refinement shellResolution: 2.5→2.565 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.335 140 -
Rwork0.263 2400 -
all-2540 -
obs--97.32 %

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