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- PDB-4wtc: CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH S15G... -

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Basic information

Entry
Database: PDB / ID: 4wtc
TitleCRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH S15G E86Q E87Q C223H V321I MUTATIONS AND DELTA8 BETA HAIRPIN LOOP DELETION IN COMPLEX WITH CDP, MN2+ AND SYMMETRICAL PRIMER TEMPLATE 5'-AGAAAUUU
Components
  • RNA PRIMER TEMPLATE AGAAAUUU
  • RNA-directed RNA polymeraseRNA-dependent RNA polymerase
KeywordsTransferase/RNA / HCV / VIRAL / NS5B / RDRP / RESISTANCE MUTATION / TEMPLATE / PRIMER / PRIMED INITIATION / DELTA8 BETA HAIRPIN LOOP DELETION / Transferase-RNA complex
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 / Dectin-2 family / 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 ...negative regulation of autophagy of mitochondrion / : / hepacivirin / host cell mitochondrial membrane / host cell lipid droplet / symbiont-mediated suppression of host TRAF-mediated signal transduction / Dectin-2 family / 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 / 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 / RNA-directed RNA polymerase / ribonucleoprotein complex / 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
CYTIDINE-5'-DIPHOSPHATE / : / RNA / Genome polyprotein
Similarity search - Component
Biological speciesHepatitis C virus JFH-1
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.75 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 1.2Mar 16, 2016Group: Data collection
Revision 1.3Sep 27, 2017Group: Derived calculations / Refinement description / Category: pdbx_struct_oper_list / software / Item: _pdbx_struct_oper_list.symmetry_operation
Revision 1.4Sep 27, 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 / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
T: RNA PRIMER TEMPLATE AGAAAUUU
P: RNA PRIMER TEMPLATE AGAAAUUU
A: RNA-directed RNA polymerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)69,4308
Polymers68,8263
Non-polymers6035
Water99155
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)141.340, 141.340, 90.800
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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RNA chain / Protein , 2 types, 3 molecules TPA

#1: RNA chain RNA PRIMER TEMPLATE AGAAAUUU


Mass: 2535.569 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Protein RNA-directed RNA polymerase / RNA-dependent 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 / Variant: S15G C223H V321I DELTA8 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q99IB8, RNA-directed RNA polymerase

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Non-polymers , 4 types, 60 molecules

#3: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mn
#4: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#5: Chemical ChemComp-CDP / CYTIDINE-5'-DIPHOSPHATE / Cytidine diphosphate


Mass: 403.176 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H15N3O11P2
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 55 / 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: 6.6
Details: NS5B AT 5.1 MG/ML IN 5 MM TRIS PH 7.5, 200 MM NH4OAC, 1 MM EDTA, 1 MM DTT AGAINST 25% PEG 550 MME, 50 MM MGCL2, 0.1 M HEPES PH 7.5 FOR CRYSTAL GROWTH SOAKED INTO 28% PEG 550 MME, 0.2 M ...Details: NS5B AT 5.1 MG/ML IN 5 MM TRIS PH 7.5, 200 MM NH4OAC, 1 MM EDTA, 1 MM DTT AGAINST 25% PEG 550 MME, 50 MM MGCL2, 0.1 M HEPES PH 7.5 FOR CRYSTAL GROWTH SOAKED INTO 28% PEG 550 MME, 0.2 M AMMONIUM ACETATE, 0.05 M BISTRIS PROPANE PH 6.0, 0.05 M TRIS PH 7.2, 6 MM MNCL2, 10 MM CDP, 4 MM 5'-AGAAAUUU WITH 8% GLYCEROL AS CRYO-PROTECTANT, CRYSTAL TRACKING ID 252991H7, UNIQUE PUCK ID YAT8-8

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-F / Wavelength: 0.97872 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Jun 12, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97872 Å / Relative weight: 1
ReflectionResolution: 2.75→50 Å / Num. obs: 26767 / % possible obs: 99.3 % / Observed criterion σ(I): -3 / Redundancy: 6.2 % / Biso Wilson estimate: 56.08 Å2 / Rmerge F obs: 0.999 / Rmerge(I) obs: 0.079 / Rrim(I) all: 0.086 / Χ2: 0.951 / Net I/σ(I): 22.23 / Num. measured all: 166026
Reflection shellResolution: 2.75→2.82 Å / Redundancy: 6.3 % / Rmerge F obs: 1 / Rmerge(I) obs: 0.727 / Mean I/σ(I) obs: 3.2 / Num. measured obs: 1567 / Num. possible: 328 / Num. unique obs: 312 / Rrim(I) all: 0.02 / Rejects: 0 / % possible all: 99.5

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Processing

Software
NameVersionClassification
XSCALEdata scaling
REFMAC5.8.0071refinement
PDB_EXTRACT3.15data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4OBC
Resolution: 2.75→50 Å / Cor.coef. Fo:Fc: 0.954 / Cor.coef. Fo:Fc free: 0.926 / SU B: 10.835 / SU ML: 0.21 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.377 / ESU R Free: 0.267 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.229 1309 4.9 %RANDOM
Rwork0.178 26766 --
obs0.181 26767 99.35 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 120.95 Å2 / Biso mean: 54.32 Å2 / Biso min: 29.2 Å2
Baniso -1Baniso -2Baniso -3
1-1.28 Å20.64 Å2-0 Å2
2--1.28 Å20 Å2
3----4.14 Å2
Refinement stepCycle: final / Resolution: 2.75→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4065 273 29 55 4422
Biso mean--47.71 45.09 -
Num. residues----548
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0194490
X-RAY DIFFRACTIONr_bond_other_d0.0010.024043
X-RAY DIFFRACTIONr_angle_refined_deg1.4541.9216183
X-RAY DIFFRACTIONr_angle_other_deg0.82639305
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.185533
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.33422.515163
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.25515655
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.8891534
X-RAY DIFFRACTIONr_chiral_restr0.0730.2706
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.0214860
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021001
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_it3.3535.4282135
X-RAY DIFFRACTIONr_mcbond_other3.3415.4272134
X-RAY DIFFRACTIONr_mcangle_it5.18.1432667
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: 2.75→2.82 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.344 110 -
Rwork0.304 1820 -
all-1930 -
obs--99.54 %

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