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- PDB-4e76: Apo crystal structure of HCV NS5B genotype 2A JFH-1 isolate with ... -

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Basic information

Entry
Database: PDB / ID: 4.0E+76
TitleApo crystal structure of HCV NS5B genotype 2A JFH-1 isolate with beta hairpin loop deletion
ComponentsRNA-directed RNA polymeraseRNA-dependent RNA polymerase
KeywordsVIRAL PROTEIN / TRANSFERASE / RDRP / loopless DELTA8 / Flaviviridae / hepatitis C virus
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
Biological speciesHepatitis C virus
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.5 Å
AuthorsEdwards, T.E. / Mosley, R.T.
CitationJournal: J.Virol. / Year: 2012
Title: Structure of hepatitis C virus polymerase in complex with primer-template RNA.
Authors: Mosley, R.T. / Edwards, T.E. / Murakami, E. / Lam, A.M. / Grice, R.L. / Du, J. / Sofia, M.J. / Furman, P.A. / Otto, M.J.
History
DepositionMar 16, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 18, 2012Provider: repository / Type: Initial release
Revision 1.1Apr 25, 2012Group: Database references
Revision 1.2Jun 6, 2012Group: Database references
Revision 1.3Jul 26, 2017Group: Refinement description / Source and taxonomy / Category: entity_src_gen / software
Revision 1.4Sep 13, 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

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Assembly

Deposited unit
A: RNA-directed RNA polymerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,4719
Polymers63,7361
Non-polymers7358
Water3,045169
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)140.690, 140.690, 92.630
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 / RNA-dependent RNA polymerase / NS5B / p68


Mass: 63736.203 Da / Num. of mol.: 1 / Fragment: SEE REMARK 999 / Mutation: E86Q, E87Q, Delta8 replaced with GG
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hepatitis C virus / Strain: JFH-1 / Gene: NS5B / Plasmid: VCID 5854 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q99IB8, RNA-directed RNA polymerase
#2: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 169 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsPROTEIN COMPRISES UNP RESIDUES 2443-3012 WITH RESIDUES 2886-2895 REPLACED WITH A GG LINKER.

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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: 4.15 Å3/Da / Density % sol: 70.38 %
Crystal growTemperature: 289 K / Method: vapor diffusion, sitting drop / pH: 6.9
Details: 3.95 mg/mL NS5B in 20 mM Tris, pH 8, 200 mM sodium chloride, 20% glycerol, 2 mM TCEP, 200 mM imidazole against 30% PEG550 MME, 0.1 M Bis-Tris propane, pH 6.5, 50 mM ammonium sulfate, ...Details: 3.95 mg/mL NS5B in 20 mM Tris, pH 8, 200 mM sodium chloride, 20% glycerol, 2 mM TCEP, 200 mM imidazole against 30% PEG550 MME, 0.1 M Bis-Tris propane, pH 6.5, 50 mM ammonium sulfate, cryoprotectant: 25% ethylene glycol, crystal tracking ID 227386E9, VAPOR DIFFUSION, SITTING DROP, temperature 289K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.3 / Wavelength: 0.9765 / Wavelength: 0.9765 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Nov 11, 2011
RadiationMonochromator: asymmetric curved crystal Si(220) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9765 Å / Relative weight: 1
ReflectionResolution: 2.5→50 Å / Num. all: 36203 / Num. obs: 34324 / % possible obs: 94.8 % / Observed criterion σ(I): -3 / Redundancy: 3.3 % / Biso Wilson estimate: 47.751 Å2 / Rmerge(I) obs: 0.078 / Net I/σ(I): 19.12
Reflection shell

Diffraction-ID: 1

Resolution (Å)Highest resolution (Å)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. unique obs% possible all
2.5-2.560.6752.328177243791.5
2.56-2.640.5732.687973239992.4
2.64-2.710.4843.237828233892.2
2.71-2.80.3953.897642226593
2.8-2.890.3264.657510224993.5
2.89-2.990.275.587162216394.5
2.99-3.10.2126.956996209894.4
3.1-3.230.1619.376812204095.8
3.23-3.370.11912.216495196795.5
3.37-3.540.08716.426213190196.9
3.54-3.730.06221.685908181196.6
3.73-3.950.04927.525587172597.7
3.95-4.230.03734.865194162297
4.23-4.560.0340.194797150996.7
4.56-50.0341.484327136996.7
5-5.590.03239.234024126097.2
5.59-6.450.03436.173433110795.7
6.45-7.910.02450.18297493596.7
7.91-11.180.01199.32239273095.8
11.180.009117.45127739991.7

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
XSCALEdata scaling
PHASERphasing
REFMACrefinement
PDB_EXTRACT3.1data extraction
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 3I5K
Resolution: 2.5→50 Å / Cor.coef. Fo:Fc: 0.943 / Cor.coef. Fo:Fc free: 0.918 / WRfactor Rfree: 0.1863 / WRfactor Rwork: 0.1507 / Occupancy max: 1 / Occupancy min: 1 / FOM work R set: 0.7625 / SU B: 19.29 / SU ML: 0.191 / SU R Cruickshank DPI: 0.297 / SU Rfree: 0.2439 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.297 / ESU R Free: 0.244 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: U VALUES : WITH TLS ADDED HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflectionSelection details
Rfree0.257 1731 5 %RANDOM
Rwork0.213 ---
obs0.215 34323 --
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 118.75 Å2 / Biso mean: 45.3948 Å2 / Biso min: 18.45 Å2
Baniso -1Baniso -2Baniso -3
1-1.82 Å20.91 Å20 Å2
2--1.82 Å20 Å2
3----2.73 Å2
Refinement stepCycle: LAST / Resolution: 2.5→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4165 0 39 169 4373
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.024293
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.6541.9745853
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.7015543
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.16422.321168
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.54515680
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.9941537
X-RAY DIFFRACTIONr_chiral_restr0.0980.2672
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.0213220
X-RAY DIFFRACTIONr_gen_planes_other
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
Rfree0.463 115 -
Rwork0.411 2266 -
all-2381 -
obs--91.3 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL112)L122)L132)L222)L232)L332)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T112)T122)T132)T222)T232)T332)Origin x (Å)Origin y (Å)Origin z (Å)
14.11030.2697-1.07441.0748-0.72995.05060.385-0.1140.5999-0.032-0.02390.1787-0.4229-0.4976-0.36110.07740.06030.06230.25630.13160.243132.7784-31.86479.285
20.70320.269-0.2210.14240.01030.8335-0.02470.21150.12380.04230.06470.00890.0783-0.2979-0.040.1127-0.0646-0.04530.33490.07720.142234.579-49.58018.0625
33.9624-0.57050.82041.50350.26570.9291-0.18620.31090.11910.06930.1976-0.17680.0082-0.1759-0.01140.1622-0.1164-0.03370.30450.04010.111350.3374-53.0508-9.9332
40.3245-0.314-0.13161.01030.3841.20050.01330.0387-0.0010.0660.04920.0190.195-0.0101-0.06250.2065-0.0323-0.04140.17470.01620.168463.4452-52.530820.8222
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A0 - 19
2X-RAY DIFFRACTION2A20 - 298
3X-RAY DIFFRACTION3A299 - 367
4X-RAY DIFFRACTION4A368 - 543

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