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- PDB-3j9b: Electron cryo-microscopy of an RNA polymerase -

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Basic information

Entry
Database: PDB / ID: 3j9b
TitleElectron cryo-microscopy of an RNA polymerase
Components
  • Polymerase basic protein 2
  • Polymerase
  • RNA (5'-R(*UP*UP*UP*UP*UP*A)-3')
  • RNA (5'-R(P*AP*AP*AP*AP*AP*A)-3')
  • RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3')
  • RNA-directed RNA polymerase catalytic subunit
KeywordsRNA BINDING PROTEIN/TRANSFERASE/RNA / Influenza RdRP / single particle reconstitution / replication / RNA BINDING PROTEIN-TRANSFERASE-RNA complex
Function / homology
Function and homology information


symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / cap snatching / viral transcription / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding ...symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / cap snatching / viral transcription / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / host cell nucleus / RNA binding / identical protein binding / metal ion binding
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / Polymerase acidic protein / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Influenza RNA-dependent RNA polymerase subunit PA
Similarity search - Domain/homology
RNA / Polymerase acidic protein / RNA-directed RNA polymerase catalytic subunit
Similarity search - Component
Biological speciesInfluenza A virus
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsChang, S.H. / Sun, D.P. / Liang, H.H. / Wang, J. / Li, J. / Guo, L. / Wang, X.L. / Guan, C.C. / Boruah, B.M. / Yuan, L.M. ...Chang, S.H. / Sun, D.P. / Liang, H.H. / Wang, J. / Li, J. / Guo, L. / Wang, X.L. / Guan, C.C. / Boruah, B.M. / Yuan, L.M. / Feng, F. / Yang, M.R. / Wojdyla, J. / Wang, J.W. / Wang, M.T. / Wang, H.W. / Liu, Y.F.
CitationJournal: Mol Cell / Year: 2015
Title: Cryo-EM structure of influenza virus RNA polymerase complex at 4.3 Å resolution.
Authors: Shenghai Chang / Dapeng Sun / Huanhuan Liang / Jia Wang / Jun Li / Lu Guo / Xiangli Wang / Chengcheng Guan / Bhargavi M Boruah / Lingmin Yuan / Feng Feng / Mingrui Yang / Lulan Wang / Yao ...Authors: Shenghai Chang / Dapeng Sun / Huanhuan Liang / Jia Wang / Jun Li / Lu Guo / Xiangli Wang / Chengcheng Guan / Bhargavi M Boruah / Lingmin Yuan / Feng Feng / Mingrui Yang / Lulan Wang / Yao Wang / Justyna Wojdyla / Lanjuan Li / Jiawei Wang / Meitian Wang / Genhong Cheng / Hong-Wei Wang / Yingfang Liu /
Abstract: Replication and transcription of influenza virus genome mainly depend on its RNA-dependent RNA polymerase (RdRP), composed of the PA, PB1, and PB2 subunits. Although extensively studied, the ...Replication and transcription of influenza virus genome mainly depend on its RNA-dependent RNA polymerase (RdRP), composed of the PA, PB1, and PB2 subunits. Although extensively studied, the underlying mechanism of the RdRP complex is still unclear. Here we report the biochemical characterization of influenza RdRP subcomplex comprising PA, PB1, and N terminus of PB2, which exist as dimer in solution and can assemble into a tetramer state, regulated by vRNA promoter. Using single-particle cryo-electron microscopy, we have reconstructed the RdRP tetramer complex at 4.3 Å, highlighting the assembly and interfaces between monomers within the tetrameric structure. The individual RdRP subcomplex contains all the characterized motifs and appears as a cage-like structure. High-throughput mutagenesis profiling revealed that residues involved in the oligomer state formation are critical for viral life cycle. Our results lay a solid base for understanding the mechanism of replication of influenza and other negative-stranded RNA viruses.
History
DepositionDec 16, 2014Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Feb 18, 2015Provider: repository / Type: Initial release
Revision 1.1Oct 9, 2019Group: Data collection / Database references ...Data collection / Database references / Other / Source and taxonomy
Category: cell / database_2 ...cell / database_2 / diffrn / em_image_scans / em_software / entity_src_gen / entity_src_nat
Item: _cell.Z_PDB / _cell.length_a ..._cell.Z_PDB / _cell.length_a / _cell.length_b / _cell.length_c / _em_software.name / _entity_src_gen.pdbx_host_org_ncbi_taxonomy_id / _entity_src_gen.pdbx_host_org_scientific_name
Revision 1.2Dec 18, 2019Group: Database references / Category: struct_ref_seq_dif / Item: _struct_ref_seq_dif.details
Revision 1.3Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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

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Structure visualization

Movie
  • Deposited structure unit
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Polymerase
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase basic protein 2
H: Polymerase
I: RNA-directed RNA polymerase catalytic subunit
J: Polymerase basic protein 2
D: RNA (5'-R(P*AP*AP*AP*AP*AP*A)-3')
E: RNA (5'-R(*UP*UP*UP*UP*UP*A)-3')
K: RNA (5'-R(P*AP*AP*AP*AP*AP*A)-3')
L: RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3')


Theoretical massNumber of molelcules
Total (without water)208,77010
Polymers208,77010
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 3 types, 6 molecules AHBICJ

#1: Protein Polymerase /


Mass: 56930.676 Da / Num. of mol.: 2 / Fragment: UNP RESIDUES 203-716
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Strain: A/goose/Guangdong/3/1997(H5N1) / Gene: PA / Cell (production host): insect cell / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q91R78
#2: Protein RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 1 / PB1 / RNA-directed RNA polymerase subunit P1


Mass: 38085.730 Da / Num. of mol.: 2 / Fragment: UNP RESIDUES 1-641
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Strain: A/Goose/Guangdong/1/1996 H5N1 genotype Gs/Gd / Gene: PB1 / Cell (production host): insect cell / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9Q0V0, RNA-directed RNA polymerase
#3: Protein Polymerase basic protein 2 / RNA-directed RNA polymerase subunit P3


Mass: 5634.938 Da / Num. of mol.: 2 / Fragment: UNP RESIDUES 1-130
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Strain: A/Goose/Guangdong/1/1996 H5N1 genotype Gs/Gd / Gene: PB2 / Cell (production host): insect cell / Production host: Trichoplusia ni (cabbage looper)

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RNA chain , 3 types, 4 molecules DKEL

#4: RNA chain RNA (5'-R(P*AP*AP*AP*AP*AP*A)-3')


Mass: 1930.277 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (natural) synthetic construct (others)
#5: RNA chain RNA (5'-R(*UP*UP*UP*UP*UP*A)-3')


Mass: 1815.077 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (natural) synthetic construct (others)
#6: RNA chain RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3')


Mass: 1792.037 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (natural) synthetic construct (others)

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Details

Sequence detailsTHE AUTHOR KNOWS THE SEQUENCE OF CHAINS A/H, B/I AND C/J, BUT IS UNSURE OF THE COORDINATES ...THE AUTHOR KNOWS THE SEQUENCE OF CHAINS A/H, B/I AND C/J, BUT IS UNSURE OF THE COORDINATES ALIGNMENT WITH THE SEQUENCE. THESE CHAINS INCLUDE MISSING RESIDUES DUE TO LACK OF ELECTRON DENSITY. THE AUTHOR STATES THAT THE ONE LETTER CODE SEQUENCES ARE AS FOLLOWS. (1) CHAIN A/H: THE SEQUENCE DATABASE IS RESIDUES 203-716 OF UNP Q91R78. ERFEITGTMCRLADQSLPPNFSSLEKFRAYVDGFEPNGCIEGKLSQMSKEVNARIE PFLK TTPRPLRLPDGPPCSQRSKFLLMDALKLSIEDPSHEGEGIPLYDAIKCMKTFFGWKEPNI VKPHEKGINPNYLLAWKQVLAELQDIENEEKIPKTKNMRKTSQLKWALGENMAPEKVDFE DCKDVSDLRQYDSDEPKPRSLASWIQSEFNKACELTDSSWIELDEIGEDVAPIEHIASMR RNYFTAEVSHCRATEYIMKGVYINTALLNASCAAMDDFQLIPMISKCRTKEGRRKTNLYG FIIKGRSHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCVLEIGDMLLRTAIGQVSRPMF LYVRANGTSKIKMKWGMEMRRCLLQSLQQIESMIEAESSVKEKDMTKEFFENKSETWPIG ESPKGMEEGSIGKVCRTLLAKSVFNSLYASPQLEGFSAESRKLLLIVQALRDNLEPGTFD LGGLYEAIEECLINDPWVLLNASWFNSFLTHALK (2) CHAIN B/I: THE SEQUENCE DATABASE IS RESIDUES 1-641 OF UNP Q9Q0V0. MDVNPTLLFLKVPAQNAISTTFPY TGDPPYSHGTGTGYTMDTVNRTHQYSEKGKWTTNTE TGAPQLNPIDGPLPEDNEPSGYAQTDCVLEAMAFLEESHPGIFENSCLETMEVVQQTRVD KLTQGRQTYDWTLKRNQPAATALANTIEVFRSNGLTANESGRLIDFLKDVMESMDKGEME IITHFQRKRRVRDNMTKKMVTQRTIGKKKQRLNKRSYLIRALTLNTMTKDAERGKLKRRA IATPGMQIRGFVYFVETLARSICEKLEQSGLPVGGNEKKAKLANVVRKMMTNSQDTELSF TITGDNTKWNENQNPRMFLAMITYITRNQPEWFRNVLSIAPIMFSNKMARLGKGYMFESK SMKLRTQIPAEMLASIDLKYFNESTRKKIEKIRPLLIDGTASLSPGMMMGMFNMLSTVLG VSILNLGQKRYTKTTYWWDGLQSSDDFALIVNAPNHEGIEAGVDRFYRTCKLVGINMTKK KSYINRTGTCEFTSFFYRYGFVANFSMELPSFGVSGINESADMSIGVTVIKNNMMDNDLG PATAQMALQLFIKDYRYPYRCHRGDTQIQTRRSFELKKLWEQTRSKAGLLVSDGGPNPYN IRNLHIPEAGLKWELMDEDYQGRLCNPLNPFVSHKEIESVN (3) CHAIN C/J: THE SEQUENCE DATABASE IS RESIDUES 1-130 OF UNP Q9Q0V1. MERIKELRDLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPALRMKWMMAMKYPITAD KRIMEMIPERNEQGQTLWSKTNDAGSDRVMVSPLAVTWWNRNGPTTSTVHYPKVYKTYFE KVERLKHGTF

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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: dimeric Influenza Virus RNA Polymerase / Type: COMPLEX
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Quantifoil Cu R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %
Method: The grids were blotted for 6.5 s with 100% humidity and flash frozen in liquid ethane cooled at liquid nitrogen temperature using an FEI Vitrobot.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Oct 2, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Cs: 2.7 mm / Camera length: 0 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1UCSF Chimeramodel fitting
2EMAN23D reconstruction
3RELION3D reconstruction
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 67066 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: REFINEMENT PROTOCOL--rigid body
Atomic model buildingPDB-ID: 3CM8

3cm8


Accession code: 3CM8 / Source name: PDB / Type: experimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms12432 422 0 0 12854

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