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- PDB-1uon: REOVIRUS POLYMERASE LAMBDA-3 LOCALIZED BY ELECTRON CRYOMICROSCOPY... -

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Entry
Database: PDB / ID: 1uon
TitleREOVIRUS POLYMERASE LAMBDA-3 LOCALIZED BY ELECTRON CRYOMICROSCOPY OF VIRIONS AT 7.6-A RESOLUTION
Components
  • 5'-R(*GP*GP*GP*GP*GP*)-3'
  • 5'-R(*UP*AP*GP*CP*CP*CP*CP*CP*)-3'
  • MINOR CORE PROTEIN LAMBDA 3
KeywordsPOLYMERASE / REOVIRUS / CRYOEM / CORE PROTEIN
Function / homology
Function and homology information


viral genome replication / viral nucleocapsid / hydrolase activity / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleotide binding / RNA binding
Similarity search - Function
Reovirus RNA-dependent RNA polymerase lambda 3 / Reovirus RNA-dependent RNA polymerase lambda 3 / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
3'-DEOXY-CYTIDINE-5'-TRIPHOSPHATE / : / RNA / RNA-directed RNA polymerase lambda-3 / RNA-directed RNA polymerase
Similarity search - Component
Biological speciesREOVIRUS
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.6 Å
AuthorsZhang, X. / Walker, S.B. / Chipman, P.R. / Nibert, M.L. / Baker, T.S.
CitationJournal: Nat Struct Biol / Year: 2003
Title: Reovirus polymerase lambda 3 localized by cryo-electron microscopy of virions at a resolution of 7.6 A.
Authors: Xing Zhang / Stephen B Walker / Paul R Chipman / Max L Nibert / Timothy S Baker /
Abstract: Reovirus is an icosahedral, double-stranded (ds) RNA virus that uses viral polymerases packaged within the viral core to transcribe its ten distinct plus-strand RNAs. To localize these polymerases, ...Reovirus is an icosahedral, double-stranded (ds) RNA virus that uses viral polymerases packaged within the viral core to transcribe its ten distinct plus-strand RNAs. To localize these polymerases, the structure of the reovirion was refined to a resolution of 7.6 A by cryo-electron microscopy (cryo-EM) and three-dimensional (3D) image reconstruction. X-ray crystal models of reovirus proteins, including polymerase lambda 3, were then fitted into the density map. Each copy of lambda 3 was found anchored to the inner surface of the icosahedral core shell, making major contacts with three molecules of shell protein lambda 1 and overlapping, but not centering on, a five-fold axis. The overlap explains why only one copy of lambda 3 is bound per vertex. lambda 3 is furthermore oriented with its transcript exit channel facing a small channel through the lambda 1 shell, suggesting how the nascent RNA is passed into the large external cavity of the pentameric capping enzyme complex formed by protein lambda 2.
History
DepositionSep 21, 2003Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 13, 2003Provider: repository / Type: Initial release
Revision 1.1May 8, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 2.0Oct 3, 2018Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Source and taxonomy
Category: atom_site / diffrn_radiation ...atom_site / diffrn_radiation / diffrn_radiation_wavelength / em_software / ndb_struct_conf_na / ndb_struct_na_base_pair / ndb_struct_na_base_pair_step / pdbx_entity_src_syn / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / struct_asym / struct_conn / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _em_software.image_processing_id / _ndb_struct_conf_na.feature / _pdbx_nonpoly_scheme.asym_id / _pdbx_nonpoly_scheme.auth_seq_num / _pdbx_nonpoly_scheme.ndb_seq_num / _pdbx_nonpoly_scheme.pdb_seq_num / _pdbx_nonpoly_scheme.pdb_strand_id / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _struct_site_gen.auth_asym_id / _struct_site_gen.auth_seq_id

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Structure visualization

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Assembly

Deposited unit
A: MINOR CORE PROTEIN LAMBDA 3
B: 5'-R(*GP*GP*GP*GP*GP*)-3'
C: 5'-R(*UP*AP*GP*CP*CP*CP*CP*CP*)-3'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)148,0788
Polymers146,5663
Non-polymers1,5115
Water6,305350
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4290 Å2
ΔGint-4.3 kcal/mol
Surface area57050 Å2
MethodPQS

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Components

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Protein , 1 types, 1 molecules A

#1: Protein MINOR CORE PROTEIN LAMBDA 3 / POLYMERASE


Mass: 142423.797 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) REOVIRUS / Strain: T3D / References: UniProt: P17378, UniProt: Q8V1A3*PLUS

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RNA chain , 2 types, 2 molecules BC

#2: RNA chain 5'-R(*GP*GP*GP*GP*GP*)-3'


Mass: 1681.072 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: RNA chain 5'-R(*UP*AP*GP*CP*CP*CP*CP*CP*)-3'


Mass: 2461.529 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 355 molecules

#4: Chemical ChemComp-CH1 / 3'-DEOXY-CYTIDINE-5'-TRIPHOSPHATE


Mass: 467.157 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C9H16N3O13P3
#5: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 350 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: REOVIRUS POLYMERASE LAMBDA-3 / Type: COMPLEX
Buffer solutionName: TRIS / pH: 7.5 / Details: TRIS
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
Crystal grow
*PLUS
Method: cryo electron microscopy / Details: cryo electron microscopy

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Electron microscopy imaging

EM imaging

Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Mode: BRIGHT FIELDBright-field microscopy / Specimen-ID: 1

IDAccelerating voltage (kV)Calibrated magnification (X)DateModelCs (mm)Nominal defocus max (nm)Nominal defocus min (nm)Nominal magnification (X)Temperature (K)Tilt angle max (°)Tilt angle min (°)
120039200Dec 1, 1997FEI/PHILIPS CM200T232001300380009700
230047440Apr 1, 2002FEI/PHILIPS CM300FEG/T45000
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector model
1120KODAK SO-163 FILM
22KODAK SO-163 FILM
Image scansNum. digital images: 54

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Processing

EM softwareName: PFT / Category: 3D reconstruction / Details: PFT2D
CTF correctionDetails: INDIVIDUAL PARTICLES, PHASE AND AMPLITUDE
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: SPHERICAL HARMONICS / Resolution: 7.6 Å / Num. of particles: 7939 / Nominal pixel size: 2.3 Å / Actual pixel size: 2.21 Å / Magnification calibration: CROSS-CORRELATION
Details: STRUCTURE OF REOVIRUS CORE (REINISCH ET AL. 2000 NATURE 404:960-967) (PDB ENTRY 1EJ6). PROTEIN ATOMS 9986, NUCLEIC ACID ATOMS 281, HETEROGEN ATOMS 86, SOLVENT ATOMS 350.
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Details: METHOD--SITUS (CHACON ET AL, 2002 J. MOL. BIOL. 317,375-394) WAS USED TO FIT THE X-RAY STRUCTURE OF REOVIRUS POLYMERASE (TAO ET AL. 2002 CELL 111, 733-745) (PDB CODE 1N35) INTO THE EM ...Details: METHOD--SITUS (CHACON ET AL, 2002 J. MOL. BIOL. 317,375-394) WAS USED TO FIT THE X-RAY STRUCTURE OF REOVIRUS POLYMERASE (TAO ET AL. 2002 CELL 111, 733-745) (PDB CODE 1N35) INTO THE EM DENSITY OF THE 7.6-A RESOLUTION MAP OF REOVIRUS VIRIONS. THE COORDINATES OF THE FITTED REOVIRUS POLYMERASE WERE ALIGNED TO THE X-RAY STRUCTURE OF REOVIRUS CORE (REINISCH ET AL. 2000 NATURE 404,960-967) (PDB ENTRY 1EJ6). ALL MATRICES FOR BUILDING AN ICOSAHEDRON CAN BE FOUND IN 1EJ6.
Atomic model buildingPDB-ID: 1UON
RefinementHighest resolution: 7.6 Å
Refinement stepCycle: LAST / Highest resolution: 7.6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9986 281 86 350 10703

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