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

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Basic information

Entry
Database: PDB / ID: 4wti
TitleCRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH S15G E86Q E87Q C223H V321I MUTATIONS IN COMPLEX WITH RNA TEMPLATE 5'-ACGG, RNA PRIMER 5'-PCC, MN2+, AND GDP
Components
  • RNA PRIMER CC
  • RNA TEMPLATE ACGG
  • RNA-directed RNA polymerase
KeywordsTransferase/RNA / HCV / VIRAL / NS5B / RDRP / RESISTANCE MUTATION / TEMPLATE / PRIMER / PRIMED INITIATION / Transferase-RNA complex
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 / 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 / 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 / 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 / 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, 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 / 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
GUANOSINE-5'-DIPHOSPHATE / : / RNA / Genome polyprotein
Similarity search - Component
Biological speciesHepatitis C virus JFH-1
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.8 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 30, 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_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.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.value / _struct_conn.pdbx_dist_value / _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
Revision 1.5Oct 30, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)67,5909
Polymers66,6653
Non-polymers9266
Water1,11762
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)140.150, 140.150, 92.760
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 , 2 types, 2 molecules TP

#1: RNA chain RNA TEMPLATE ACGG


Mass: 1279.842 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: RNA chain RNA PRIMER CC


Mass: 565.406 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein , 1 types, 1 molecules A

#3: Protein RNA-directed RNA polymerase


Mass: 64819.379 Da / Num. of mol.: 1 / Mutation: S2457G, E2528Q, E2529Q, C2665H, V2763I
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

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Non-polymers , 5 types, 68 molecules

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


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


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#6: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#7: Chemical ChemComp-B3P / 2-[3-(2-HYDROXY-1,1-DIHYDROXYMETHYL-ETHYLAMINO)-PROPYLAMINO]-2-HYDROXYMETHYL-PROPANE-1,3-DIOL


Mass: 282.334 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H26N2O6 / Comment: pH buffer*YM
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 62 / Source method: isolated from a natural source / Formula: H2O

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Details

Has protein modificationY

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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: 4.1 Å3/Da / Density % sol: 70 %
Crystal growTemperature: 289 K / Method: vapor diffusion, sitting drop / pH: 6.6
Details: NS5B AT 4.7 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 4.7 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 GDP, 2 MM 5'-ACGG, 2 MM 5'-PCC, WITH 8% GLYCEROL AS CRYO-PROTECTANT, CRYSTAL TRACKING ID 251148A1, UNIQUE PUCK ID HDW8-2

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-G / Wavelength: 0.97872 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Dec 12, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97872 Å / Relative weight: 1
ReflectionResolution: 2.8→50 Å / Num. obs: 25574 / % possible obs: 99.7 % / Observed criterion σ(I): -3 / Redundancy: 6.1 % / Biso Wilson estimate: 59.52 Å2 / Rmerge F obs: 0.998 / Rmerge(I) obs: 0.076 / Rrim(I) all: 0.083 / Χ2: 0.975 / Net I/σ(I): 19.4 / Num. measured all: 154849
Reflection shellResolution: 2.8→2.87 Å / Redundancy: 6.2 % / Rmerge F obs: 0.999 / Rmerge(I) obs: 0.695 / Mean I/σ(I) obs: 2.5 / Num. measured obs: 1360 / Num. possible: 311 / Num. unique obs: 276 / Rrim(I) all: 0.028 / Rejects: 0 / % possible all: 99.9

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Processing

Software
NameVersionClassification
XSCALEdata scaling
REFMAC5.8.0049refinement
PDB_EXTRACT3.15data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 4OBC

4obc


Resolution: 2.8→50 Å / Cor.coef. Fo:Fc: 0.955 / Cor.coef. Fo:Fc free: 0.928 / SU B: 11.668 / SU ML: 0.222 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.43 / ESU R Free: 0.286 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.234 1303 5.1 %RANDOM
Rwork0.181 25570 --
obs0.184 25574 99.72 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 135.56 Å2 / Biso mean: 61.5 Å2 / Biso min: 37.34 Å2
Baniso -1Baniso -2Baniso -3
1-1.77 Å20.88 Å2-0 Å2
2--1.77 Å20 Å2
3----5.74 Å2
Refinement stepCycle: final / Resolution: 2.8→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4219 108 92 62 4481
Biso mean--81.47 51.95 -
Num. residues----557
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0194501
X-RAY DIFFRACTIONr_bond_other_d0.0010.024151
X-RAY DIFFRACTIONr_angle_refined_deg1.381.9556167
X-RAY DIFFRACTIONr_angle_other_deg0.96839529
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.0355556
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.55622.281171
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.49315682
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.6251537
X-RAY DIFFRACTIONr_chiral_restr0.0740.2703
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.0214988
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021032
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_it3.346.222215
X-RAY DIFFRACTIONr_mcbond_other3.3356.2182214
X-RAY DIFFRACTIONr_mcangle_it5.1269.3242768
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: 2.8→2.87 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.436 97 -
Rwork0.341 1756 -
all-1853 -
obs--99.89 %

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