[English] 日本語
Yorodumi
- PDB-7orn: La Crosse virus polymerase at replication initiation stage -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 7orn
TitleLa Crosse virus polymerase at replication initiation stage
Components
  • La Crosse virus polymerase
  • RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*UP*AP*CP*CP*AP*AP*G)-3')
  • RNA (5'-R(P*AP*CP*UP*UP*GP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
KeywordsVIRAL PROTEIN / RNA-dependent RNA polymerase
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 / nucleotide binding / RNA-directed RNA polymerase activity ...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 / nucleotide binding / RNA-directed RNA polymerase activity / DNA-templated transcription / RNA binding / metal ion binding
Similarity search - Function
Restriction Endonuclease - #60 / 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 / Restriction Endonuclease ...Restriction Endonuclease - #60 / 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 / Restriction Endonuclease / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / RNA / RNA (> 10) / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesLa Crosse orthobunyavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsArragain, B. / Durieux Trouilleton, Q. / Baudin, F. / Cusack, S. / Schoehn, G. / Malet, H.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-19-CE11-0024-02 France
CitationJournal: Nat Commun / Year: 2022
Title: Structural snapshots of La Crosse virus polymerase reveal the mechanisms underlying Peribunyaviridae replication and transcription.
Authors: Benoît Arragain / Quentin Durieux Trouilleton / Florence Baudin / Jan Provaznik / Nayara Azevedo / Stephen Cusack / Guy Schoehn / Hélène Malet /
Abstract: Segmented negative-strand RNA bunyaviruses encode a multi-functional polymerase that performs genome replication and transcription. Here, we establish conditions for in vitro activity of La Crosse ...Segmented negative-strand RNA bunyaviruses encode a multi-functional polymerase that performs genome replication and transcription. Here, we establish conditions for in vitro activity of La Crosse virus polymerase and visualize its conformational dynamics by cryo-electron microscopy, unveiling the precise molecular mechanics underlying its essential activities. We find that replication initiation is coupled to distal duplex promoter formation, endonuclease movement, prime-and-realign loop extension and closure of the polymerase core that direct the template towards the active site. Transcription initiation depends on C-terminal region closure and endonuclease movements that prompt primer cleavage prior to primer entry in the active site. Product realignment after priming, observed in replication and transcription, is triggered by the prime-and-realign loop. Switch to elongation results in polymerase reorganization and core region opening to facilitate template-product duplex formation in the active site cavity. The uncovered detailed mechanics should be helpful for the future design of antivirals counteracting bunyaviral life threatening pathogens.
History
DepositionJun 6, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 16, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 2, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-13043
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: La Crosse virus polymerase
H: RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*UP*AP*CP*CP*AP*AP*G)-3')
T: RNA (5'-R(P*AP*CP*UP*UP*GP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)278,6566
Polymers278,1003
Non-polymers5563
Water1,65792
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9370 Å2
ΔGint-85 kcal/mol
Surface area67500 Å2
MethodPISA

-
Components

-
Protein , 1 types, 1 molecules A

#1: Protein La Crosse virus polymerase


Mass: 264751.062 Da / Num. of mol.: 1 / Mutation: H34K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) La Crosse orthobunyavirus / Gene: L segment / Cell line (production host): Hi 5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A5HC98, RNA-directed RNA polymerase

-
RNA chain , 2 types, 2 molecules HT

#2: RNA chain RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*UP*AP*CP*CP*AP*AP*G)-3')


Mass: 5466.325 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Mutated 5' sequence of La Crosse virus M segment Nucleotides G2, U3, A9 and C10 were mutated into C2, G3, C9 and G10
Source: (synth.) La Crosse orthobunyavirus
#3: RNA chain RNA (5'-R(P*AP*CP*UP*UP*GP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')


Mass: 7882.668 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: 3' extremity (25 nucleotides) of La Crosse virus M segment
Source: (synth.) La Crosse orthobunyavirus

-
Non-polymers , 3 types, 95 molecules

#4: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water


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

-
Details

Has ligand of interestY

-
Experimental details

-
Experiment

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

-
Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1La Crosse virus polymerase at replication initiationCOMPLEX#1-#30RECOMBINANT
2La Crosse virus polymeraseCOMPLEX#11RECOMBINANT
3RNACOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 0.279 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22La Crosse orthobunyavirus2560547
33La Crosse orthobunyavirus2560547
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Trichoplusia ni (cabbage looper)7111
23synthetic construct (others)32630
Buffer solutionpH: 8
Details: 50 mM TRIS-HCl pH 8, 150 mM NaCl, 5 mM BME, 100 uM ATP/UTP and 5mM MgCl2.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-Hcl1
2150 mMNaClNaCl1
35 mMbeta-mercaptoethanol1
4100 uMATP1
5100 uMUTP1
65 mMMgCl21
SpecimenConc.: 0.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 1.3 uM LACV-LCItag_H34K were sequentially incubated for 1h at 4 degree with (i) 1.9 uM 5prime 1-17 BPm, (ii) 1.9 uM 3prime vRNA 1-25. 100 uM ATP/UTP and 5mM MgCl2 for 4h at 30dregree
Specimen supportDetails: 25 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: blot force 1 2 sec

-
Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Calibrated magnification: 130000 X / Nominal defocus max: 18000 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 18000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 15573
EM imaging opticsEnergyfilter name: GIF Bioquantum
Image scansWidth: 5760 / Height: 4092

-
Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3particle selectionBlob picker
2SerialEMimage acquisition
4cryoSPARC3CTF correctionPatch CTF correction
7Coot0.9.2model fitting
9cryoSPARC3initial Euler assignment
10RELION3.1final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.19.2model refinementreal space refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4615689
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 641008 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 64.15 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6Z6G

6z6g


Pdb chain-ID: A / Accession code: 6Z6G / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315074
ELECTRON MICROSCOPYf_angle_d0.47520510
ELECTRON MICROSCOPYf_dihedral_angle_d7.1032279
ELECTRON MICROSCOPYf_chiral_restr0.0412307
ELECTRON MICROSCOPYf_plane_restr0.0032460

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more