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- PDB-4wt9: APO CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH ... -

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Basic information

Entry
Database: PDB / ID: 4wt9
TitleAPO CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH E86Q E87Q S15G C223H V321I AND DELTA8 MUTATIONS
ComponentsRNA-directed RNA polymerase
KeywordsTRANSFERASE / HCV / VIRAL / NS5B / RDRP / RESISTANCE MUTATION / DELTA8 BETA HAIRPIN LOOP DELETION
Function / homology
Function and homology information


hepacivirin / host cell mitochondrial membrane / host cell lipid droplet / symbiont-mediated suppression of host TRAF-mediated signal transduction / symbiont-mediated transformation of host cell / 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 / SH3 domain binding ...hepacivirin / host cell mitochondrial membrane / host cell lipid droplet / symbiont-mediated suppression of host TRAF-mediated signal transduction / symbiont-mediated transformation of host cell / 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 / SH3 domain binding / nucleoside-triphosphate phosphatase / channel activity / viral nucleocapsid / monoatomic ion transmembrane transport / 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 / ribonucleoprotein complex / symbiont-mediated activation of host autophagy / RNA-directed RNA polymerase / serine-type endopeptidase activity / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-directed RNA polymerase activity / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / identical protein 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, 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 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 core protein, chain A superfamily / : / 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 / NS3 RNA helicase, C-terminal helical domain / 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 / Resolution: 2.5 Å
AuthorsEdwards, T.E. / Appleby, T.C.
CitationJournal: Science / Year: 2015
Title: Structural basis for RNA replication by the hepatitis C virus polymerase.
Authors: Appleby, T.C. / Perry, J.K. / Murakami, E. / Barauskas, O. / Feng, J. / Cho, A. / Fox, D. / Wetmore, D.R. / McGrath, M.E. / Ray, A.S. / Sofia, M.J. / Swaminathan, S. / Edwards, T.E.
History
DepositionOct 29, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 11, 2015Provider: repository / Type: Initial release
Revision 1.1Feb 25, 2015Group: Database references
Revision 1.2Sep 27, 2017Group: Database references / Derived calculations ...Database references / Derived calculations / Other / Refinement description / Source and taxonomy / Structure summary
Category: citation / entity_src_gen ...citation / entity_src_gen / pdbx_database_status / pdbx_struct_assembly / pdbx_struct_oper_list / software / struct_keywords
Item: _citation.journal_id_CSD / _entity_src_gen.pdbx_alt_source_flag ..._citation.journal_id_CSD / _entity_src_gen.pdbx_alt_source_flag / _pdbx_database_status.pdb_format_compatible / _pdbx_struct_assembly.oligomeric_details / _pdbx_struct_oper_list.symmetry_operation / _struct_keywords.pdbx_keywords
Revision 1.3Sep 27, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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)64,2954
Polymers63,7551
Non-polymers5403
Water3,657203
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)140.370, 140.370, 92.230
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number170
Space group name H-MP65

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Components

#1: Protein RNA-directed RNA polymerase


Mass: 63755.211 Da / Num. of mol.: 1
Mutation: S2457G, E2528Q, E2529Q, C2665H, V2763I, UNP residues 2886-2895 NFEMYGSVYS deleted and replaced by GG linker
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hepatitis C virus JFH-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q99IB8, RNA-directed RNA polymerase
#2: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#3: Chemical ChemComp-EPE / 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID / HEPES


Mass: 238.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18N2O4S / Comment: pH buffer*YM
#4: Chemical ChemComp-PG6 / 1-(2-METHOXY-ETHOXY)-2-{2-[2-(2-METHOXY-ETHOXY]-ETHOXY}-ETHANE


Mass: 266.331 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C12H26O6
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 203 / 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 Å3/Da / Density % sol: 70 %
Crystal growTemperature: 289 K / Method: vapor diffusion, sitting drop / pH: 6.9
Details: NS5B AT 4.08 MG/ML IN 20 MM TRIS PH 8, 200 MM NACL, 20% GLYCEROL, 2 MM TCEP, 200 MM IMIDAZOLE AGAINST 30% PEG 550 MME, 0.1 M HEPES PH 7.5, 50 MM MAGNESIUM CHLORIDE WITH 15% ETHYLENE GLYCOL ...Details: NS5B AT 4.08 MG/ML IN 20 MM TRIS PH 8, 200 MM NACL, 20% GLYCEROL, 2 MM TCEP, 200 MM IMIDAZOLE AGAINST 30% PEG 550 MME, 0.1 M HEPES PH 7.5, 50 MM MAGNESIUM CHLORIDE WITH 15% ETHYLENE GLYCOL AS CRYO-PROTECTANT, CRYSTAL TRACKING ID 232769E7

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-D / Wavelength: 1.07809 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Mar 28, 2012
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.07809 Å / Relative weight: 1
ReflectionResolution: 2.5→50 Å / Num. obs: 35345 / % possible obs: 98.5 % / Observed criterion σ(I): -3 / Redundancy: 3.6 % / Biso Wilson estimate: 46.96 Å2 / Rmerge F obs: 0.158 / Rmerge(I) obs: 0.084 / Rrim(I) all: 0.099 / Χ2: 0.973 / Net I/σ(I): 16.62 / Num. measured all: 127140
Reflection shellResolution: 2.5→2.56 Å / Redundancy: 3.7 % / Rmerge F obs: 0.016 / Rmerge(I) obs: 0.706 / Mean I/σ(I) obs: 2.6 / Num. measured obs: 1268 / Num. possible: 426 / Num. unique obs: 394 / Rrim(I) all: 0.021 / Rejects: 0 / % possible all: 98.6

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Processing

Software
NameVersionClassification
XSCALEdata scaling
REFMAC5.6.0117refinement
PDB_EXTRACT3.15data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 4E76

4e76


Resolution: 2.5→50 Å / Cor.coef. Fo:Fc: 0.951 / Cor.coef. Fo:Fc free: 0.918 / SU B: 8.197 / SU ML: 0.171 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.258 / ESU R Free: 0.227 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.245 1774 5 %RANDOM
Rwork0.193 35344 --
obs0.195 35345 98.5 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 102.63 Å2 / Biso mean: 33.09 Å2 / Biso min: 14.72 Å2
Baniso -1Baniso -2Baniso -3
1-1.74 Å20.87 Å20 Å2
2--1.74 Å20 Å2
3----2.61 Å2
Refinement stepCycle: final / Resolution: 2.5→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4165 0 31 203 4399
Biso mean--62.88 35.91 -
Num. residues----544
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.024291
X-RAY DIFFRACTIONr_bond_other_d0.0010.022918
X-RAY DIFFRACTIONr_angle_refined_deg1.5291.9715838
X-RAY DIFFRACTIONr_angle_other_deg0.91737094
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2875542
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.60122.335167
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.11615682
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.8291536
X-RAY DIFFRACTIONr_chiral_restr0.080.2666
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.0214737
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02883
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_it
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it
X-RAY DIFFRACTIONr_scbond_it
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.5→2.56 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.473 123 -
Rwork0.338 2421 -
all-2544 -
obs--98.53 %

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