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- PDB-5amr: Structure of the La Crosse Bunyavirus polymerase in complex with ... -

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Basic information

Entry
Database: PDB / ID: 5amr
TitleStructure of the La Crosse Bunyavirus polymerase in complex with the 3' viral RNA
Components
  • (RNA) x 2
  • RNA POLYMERASE L
KeywordsHYDROLASE / POLYMERASE / RNADRNAPOL / BUNYAVIRUS / RNA
Function / homology
Function and homology information


host cell endoplasmic reticulum / virion component / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell Golgi apparatus / Hydrolases; Acting on ester bonds / hydrolase activity / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding ...host cell endoplasmic reticulum / virion component / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell Golgi apparatus / Hydrolases; Acting on ester bonds / hydrolase activity / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / RNA binding / metal ion binding
Similarity search - Function
RNA-directed RNA polymerase, orthobunyavirus / : / Virus, RNA-directed RNA polymerase L, thumb ring domain / RNA-directed RNA polymerase L, N-terminal / L protein N-terminus / : / RNA-dependent RNA polymerase, bunyaviral / Bunyavirus RNA dependent RNA polymerase / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile.
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesBUNYAVIRUS LA CROSSE
MethodX-RAY DIFFRACTION / SYNCHROTRON / MIRAS / Resolution: 2.57 Å
AuthorsReguera, J. / Gerlach, P. / Cusack, S.
CitationJournal: Cell / Year: 2015
Title: Structural Insights into Bunyavirus Replication and Its Regulation by the vRNA Promoter.
Authors: Piotr Gerlach / Hélène Malet / Stephen Cusack / Juan Reguera /
Abstract: Segmented negative-strand RNA virus (sNSV) polymerases transcribe and replicate the viral RNA (vRNA) within a ribonucleoprotein particle (RNP). We present cryo-EM and X-ray structures of, ...Segmented negative-strand RNA virus (sNSV) polymerases transcribe and replicate the viral RNA (vRNA) within a ribonucleoprotein particle (RNP). We present cryo-EM and X-ray structures of, respectively, apo- and vRNA bound La Crosse orthobunyavirus (LACV) polymerase that give atomic-resolution insight into how such RNPs perform RNA synthesis. The complementary 3' and 5' vRNA extremities are sequence specifically bound in separate sites on the polymerase. The 5' end binds as a stem-loop, allosterically structuring functionally important polymerase active site loops. Identification of distinct template and product exit tunnels allows proposal of a detailed model for template-directed replication with minimal disruption to the circularised RNP. The similar overall architecture and vRNA binding of monomeric LACV to heterotrimeric influenza polymerase, despite high sequence divergence, suggests that all sNSV polymerases have a common evolutionary origin and mechanism of RNA synthesis. These results will aid development of replication inhibitors of diverse, serious human pathogenic viruses.
History
DepositionMar 12, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 3, 2015Provider: repository / Type: Initial release
Revision 1.1Jun 17, 2015Group: Database references
Revision 1.2Apr 3, 2019Group: Data collection / Other / Source and taxonomy
Category: entity_src_gen / pdbx_database_proc / pdbx_database_status
Item: _entity_src_gen.pdbx_host_org_cell_line / _pdbx_database_status.recvd_author_approval

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA POLYMERASE L
B: RNA
C: RNA


Theoretical massNumber of molelcules
Total (without water)270,9993
Polymers270,9993
Non-polymers00
Water1,65792
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4560 Å2
ΔGint-25.3 kcal/mol
Surface area74410 Å2
MethodPISA
Unit cell
Length a, b, c (Å)102.000, 140.700, 162.400
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein RNA POLYMERASE L


Mass: 263429.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) BUNYAVIRUS LA CROSSE / Plasmid: PFASTBAC / Cell line (production host): High Five / Production host: TRICHOPLUSIA NI (cabbage looper) / References: UniProt: A5HC98, RNA-directed RNA polymerase
#2: RNA chain RNA /


Mass: 5060.023 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) BUNYAVIRUS LA CROSSE / Plasmid: PFASTBAC / Cell line (production host): High Five / Production host: TRICHOPLUSIA NI (cabbage looper)
#3: RNA chain RNA /


Mass: 2509.577 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) BUNYAVIRUS LA CROSSE / Plasmid: PFASTBAC / Cell line (production host): High Five / Production host: TRICHOPLUSIA NI (cabbage looper)
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 92 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsMDYQEYQQFLARINTARDACVAKDIDVDLLMARHDYFG RELCKSLNIEYRNDVPFIDIILDIRPEVDPLTIDAPHITPDNYLYINNVLYIIDYKVSVS NESSVIT

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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: 2.9 Å3/Da / Density % sol: 62 % / Description: NONE
Crystal growpH: 7
Details: 0.1M HEPES PH 7, 0.2M SODIUM CITRATE DIHYDRATE, 0.3M AMMONIUM SULPHATE, 15% PEG 3350.

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Data collection

DiffractionMean temperature: 77 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-1 / Wavelength: 0.99187
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jul 27, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.99187 Å / Relative weight: 1
ReflectionResolution: 2.57→50 Å / Num. obs: 71095 / % possible obs: 94.7 % / Observed criterion σ(I): 3 / Redundancy: 3.55 % / Rmerge(I) obs: 0.01 / Net I/σ(I): 11.2
Reflection shellResolution: 2.57→2.6 Å / Redundancy: 3.56 % / Rmerge(I) obs: 0.09 / Mean I/σ(I) obs: 1.28 / % possible all: 97.4

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Processing

Software
NameVersionClassification
PHENIX(PHENIX.REFINE)refinement
XDSdata reduction
XSCALEdata scaling
SHARPphasing
RefinementMethod to determine structure: MIRAS
Starting model: NONE

Resolution: 2.57→48.657 Å / SU ML: 0.43 / σ(F): 1.35 / Phase error: 28.76 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2523 6809 5 %
Rwork0.2038 --
obs0.2062 137111 95.34 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.57→48.657 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13571 500 0 92 14163
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00314398
X-RAY DIFFRACTIONf_angle_d0.63419512
X-RAY DIFFRACTIONf_dihedral_angle_d12.9985543
X-RAY DIFFRACTIONf_chiral_restr0.0342193
X-RAY DIFFRACTIONf_plane_restr0.0022374
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.57-2.59920.4241940.36584577X-RAY DIFFRACTION98
2.5992-2.62980.37282120.35184399X-RAY DIFFRACTION97
2.6298-2.66190.35122540.34594435X-RAY DIFFRACTION97
2.6619-2.69560.34342450.32864348X-RAY DIFFRACTION97
2.6956-2.7310.34562640.31334357X-RAY DIFFRACTION97
2.731-2.76840.3252460.30024374X-RAY DIFFRACTION95
2.7684-2.8080.35522750.29794303X-RAY DIFFRACTION96
2.808-2.84990.40342040.2974455X-RAY DIFFRACTION97
2.8499-2.89440.33472250.28614442X-RAY DIFFRACTION98
2.8944-2.94190.32772290.26774406X-RAY DIFFRACTION97
2.9419-2.99260.29152230.26524493X-RAY DIFFRACTION97
2.9926-3.0470.33832470.25454357X-RAY DIFFRACTION97
3.047-3.10560.29721970.25094366X-RAY DIFFRACTION96
3.1056-3.1690.3242170.24374426X-RAY DIFFRACTION96
3.169-3.23790.33132050.25514387X-RAY DIFFRACTION96
3.2379-3.31320.29052520.24574318X-RAY DIFFRACTION95
3.3132-3.3960.3032450.22994332X-RAY DIFFRACTION94
3.396-3.48780.26532240.21214328X-RAY DIFFRACTION96
3.4878-3.59040.27922520.20274360X-RAY DIFFRACTION96
3.5904-3.70620.24622140.18974361X-RAY DIFFRACTION95
3.7062-3.83860.24782180.19124340X-RAY DIFFRACTION95
3.8386-3.99230.25432290.17224259X-RAY DIFFRACTION94
3.9923-4.17390.19122090.16364352X-RAY DIFFRACTION95
4.1739-4.39380.20832580.15674179X-RAY DIFFRACTION93
4.3938-4.66890.20042370.14964263X-RAY DIFFRACTION93
4.6689-5.0290.20051990.15184270X-RAY DIFFRACTION94
5.029-5.53450.19932140.16374248X-RAY DIFFRACTION94
5.5345-6.33380.26171920.18534271X-RAY DIFFRACTION93
6.3338-7.97430.21461930.18164212X-RAY DIFFRACTION92
7.9743-48.66580.17242360.16024084X-RAY DIFFRACTION90

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