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- PDB-2fm2: HCV NS3-4A protease domain complexed with a ketoamide inhibitor, ... -

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Basic information

Entry
Database: PDB / ID: 2fm2
TitleHCV NS3-4A protease domain complexed with a ketoamide inhibitor, SCH446211
Components
  • NS3 protease/helicase
  • NS4a protein
KeywordsHYDROLASE / Hepatitis C Virus / HCV / NS3/4A protease / ketoamide inhibitor
Function / homology
Function and homology information


host cell mitochondrial membrane / host cell lipid droplet / symbiont-mediated suppression of host TRAF-mediated signal transduction / 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 / ribonucleoside triphosphate phosphatase activity / lipid droplet / protein complex oligomerization ...host cell mitochondrial membrane / host cell lipid droplet / symbiont-mediated suppression of host TRAF-mediated signal transduction / 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 / ribonucleoside triphosphate phosphatase activity / lipid droplet / protein complex oligomerization / monoatomic ion channel activity / viral nucleocapsid / clathrin-dependent endocytosis of virus by host cell / RNA helicase activity / host cell perinuclear region of cytoplasm / host cell endoplasmic reticulum membrane / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / ribonucleoprotein complex / induction by virus of host autophagy / virus-mediated perturbation of host defense response / 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 / virion attachment to host cell / host cell plasma membrane / virion membrane / structural molecule activity / proteolysis / RNA binding / zinc ion binding / ATP binding / plasma membrane / cytoplasm
Similarity search - Function
Thrombin, subunit H - #120 / : / 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 ...Thrombin, subunit H - #120 / : / 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 / helicase superfamily c-terminal domain / Trypsin-like serine proteases / 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 / Thrombin, subunit H / 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 / Beta Barrel / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta
Similarity search - Domain/homology
TERT-BUTYL / BETA-MERCAPTOETHANOL / : / Genome polyprotein
Similarity search - Component
Biological speciesHepatitis C virus
MethodX-RAY DIFFRACTION / FOURIER SYNTHESIS / Resolution: 2.7 Å
AuthorsYi, M. / Tong, X. / Skelton, A. / Chase, R. / Chen, T. / Prongay, A. / Bogen, S.L. / Saksena, A.K. / Njoroge, F.G. / Veselenak, R.L. ...Yi, M. / Tong, X. / Skelton, A. / Chase, R. / Chen, T. / Prongay, A. / Bogen, S.L. / Saksena, A.K. / Njoroge, F.G. / Veselenak, R.L. / Pyles, R.B. / Bourne, N. / Malcolm, B.A. / Lemon, S.M.
CitationJournal: J.Biol.Chem. / Year: 2006
Title: Mutations conferring resistance to SCH6, a novel hepatitis C virus NS3/4A protease inhibitor. Reduced RNA replication fitness and partial rescue by second-site mutations
Authors: Yi, M. / Tong, X. / Skelton, A. / Chase, R. / Chen, T. / Prongay, A. / Bogen, S.L. / Saksena, A.K. / Njoroge, F.G. / Veselenak, R.L. / Pyles, R.B. / Bourne, N. / Malcolm, B.A. / Lemon, S.M.
History
DepositionJan 6, 2006Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 7, 2006Provider: repository / Type: Initial release
Revision 1.1May 1, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Dec 21, 2022Group: Database references / Derived calculations / Structure summary
Category: chem_comp / database_2 ...chem_comp / database_2 / entity / pdbx_entity_nonpoly / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _chem_comp.name / _database_2.pdbx_DOI ..._chem_comp.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _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_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.pdbx_leaving_atom_flag / _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_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 / _struct_conn.ptnr2_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Apr 3, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Remark 300BIOMOLECULE: 1 THIS ENTRY CONTAINS THE CRYSTALLOGRAPHIC ASYMMETRIC UNIT WHICH CONSISTS OF 4 CHAIN(S) ...BIOMOLECULE: 1 THIS ENTRY CONTAINS THE CRYSTALLOGRAPHIC ASYMMETRIC UNIT WHICH CONSISTS OF 4 CHAIN(S).THE ASYMMETRIC UNIT CONTAINS A DIMER OF THE PROTEASE DOMAIN WHICH IS THE N-TERMINAL 181 RESIDUES OF THE NS3 PROTEIN (631 RESIDUES) THAT CONTAINS BOTH THE PROTEASE DOMAIN AND THE HELICASE DOMAIN. BIOLOGICALLY THE PROTEASE DOMAIN FUNCTIONS AS PART OF THE LARGER (631 RESIDUE) PROTEIN. THE LARGER PROTEIN HAS NOT BEEN SHOWN TO EXIST AS A DIMER. THE SMALLER PROTEASE DOMAIN (RESIDUES 1-181) THAT HAVE BEEN EXPRESSED WITH AN N-TERMINAL T7 EPITOPE TAG AND A C-TERMINAL HIS TAG, EXISTS AS A DIMER IN THE ASYMMETRIC UNIT. IT IS UNKNOWN WHETHER THIS PROTEIN DOMAIN IS FUNCTIONAL AS A MONOMER OR A DIMER.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: NS3 protease/helicase
B: NS4a protein
C: NS3 protease/helicase
D: NS4a protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,1888
Polymers47,2554
Non-polymers9344
Water2,774154
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7980 Å2
ΔGint-130 kcal/mol
Surface area15330 Å2
MethodPISA
Unit cell
Length a, b, c (Å)224.909, 224.909, 75.402
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number155
Space group name H-MH32
DetailsThe asymmetric unit contains a dimer of the protease domain which is the N-terminal 181 residues of the NS3 protein (631 residues) that contains both the protease domain and the helicase domain. Biological the protease domain functions as part of the larger (631 residue) protein. The larger protein has not been shown to exist as a dimer. The smaller protease domain (residues 1-181) that have been expressed with an N-terminal T7 epitope tag and a C-terminal His Tag, exists as a dimer in the asymmetric unit. This protein domain is believed to be functional as either a monomer or a dimer.

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Components

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Protein / Protein/peptide , 2 types, 4 molecules ACBD

#1: Protein NS3 protease/helicase


Mass: 21233.225 Da / Num. of mol.: 2 / Fragment: protease domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hepatitis C virus / Genus: Hepacivirus / Strain: H1A / Gene: NS polyprotein / Plasmid: pET-3A / Production host: Escherichia coli (E. coli) / Strain (production host): BLR(DE3) / References: GenBank: 28921568, UniProt: Q9ELS8*PLUS
#2: Protein/peptide NS4a protein /


Mass: 2394.039 Da / Num. of mol.: 2
Fragment: residues 24-39 with 2 LYS at both C and N-terminal
Source method: obtained synthetically
Details: Peptides were synthesized using Fmoc solid-phase chemistry on an ABI 431 synthesizer
References: UniProt: Q9ELS8*PLUS

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Non-polymers , 4 types, 158 molecules

#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-BME / BETA-MERCAPTOETHANOL / 2-Mercaptoethanol


Mass: 78.133 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6OS
#5: Chemical ChemComp-3BC / TERT-BUTYL [(1S)-1-({(1R,2S,5S)-2-[(3S,10S)-3-(CYCLOPROPYLMETHYL)-12-METHYL-4,5,8,11-TETRAOXO-10-PHENYL-2,6,9,12-TETRAAZATRIDECAN-1 -OYL]-6,6-DIMETHYL-3-AZABICYCLO[3.1.0]HEX-3-YL}CARBONYL)-2,2-DIMETHYLPROPYL]CARBAMATE / N-(R-CARBOXY-ETHYL)-ALPHA-(S)-(2-PHENYLETHYL)GLYCYL-L-ARGININE-N-PHENYLAMIDE / Butyl group


Mass: 724.887 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C38H56N6O8
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 154 / 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: 3.88 Å3/Da / Density % sol: 68.31 %
Crystal growTemperature: 285 K / Method: vapor diffusion, hanging drop / pH: 5.7
Details: Crystallization was performed by the vapor diffusion method using hanging drops (4 uL protein solution mixed with 4 uL (0.75 - 1.00)M NaCl - 0.1M MES - 0.1M Na/K PO4, pH 5.6 - 5.8) suspended ...Details: Crystallization was performed by the vapor diffusion method using hanging drops (4 uL protein solution mixed with 4 uL (0.75 - 1.00)M NaCl - 0.1M MES - 0.1M Na/K PO4, pH 5.6 - 5.8) suspended over 1mL reservoir solutions of (1.25 - 1.50)M NaCl - 0.1M MES - 0.1M Na/K PO4 - 5mM-mercaptoethanol, pH 5.6-5.8. The trays were set at 4C for 5-7 days to control nucleation, followed by incubation for 3 weeks at 12C to maximize crystal growth. , pH 5.7, VAPOR DIFFUSION, HANGING DROP, temperature 285K

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Data collection

DiffractionMean temperature: 95 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RUH3R / Wavelength: 1.5418 Å
DetectorType: RIGAKU RAXIS IV / Detector: IMAGE PLATE / Date: Apr 14, 2005 / Details: MULTILAYER X-RAY FOCUSING OPTICS
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.7→50 Å / Num. all: 19889 / Num. obs: 19511 / % possible obs: 98.1 % / Observed criterion σ(F): 1.7 / Observed criterion σ(I): 3 / Redundancy: 4 % / Biso Wilson estimate: 32.938 Å2 / Rsym value: 0.088 / Net I/σ(I): 13.6
Reflection shellResolution: 2.7→2.78 Å / Redundancy: 2.6 % / Mean I/σ(I) obs: 3 / Num. unique all: 1636 / Rsym value: 0.323 / % possible all: 84.7

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Processing

Software
NameVersionClassification
HKL-2000data collection
SCALEPACKdata scaling
X-PLORmodel building
X-PLOR98.1refinement
HKL-2000data reduction
X-PLORphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: HCV NS3(139A)/4A protease domain structure
Resolution: 2.7→8 Å / σ(F): 1.7 / σ(I): 3 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.26 1713 -RANDOM
Rwork0.185 ---
all0.189 19093 --
obs0.189 17354 98 %-
Refinement stepCycle: LAST / Resolution: 2.7→8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2714 0 58 154 2926
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONx_bond_d0.008
X-RAY DIFFRACTIONx_angle_deg1.93
X-RAY DIFFRACTIONx_improper_angle_d1.537
LS refinement shellResolution: 2.7→2.82 Å
RfactorNum. reflection% reflection
Rfree0.3493 147 -
Rwork0.3081 --
obs-1230 51.29 %

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