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- PDB-6z6b: Structure of full-length La Crosse virus L protein (polymerase) -

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Basic information

Entry
Database: PDB / ID: 6z6b
TitleStructure of full-length La Crosse virus L protein (polymerase)
Components
  • RNA (5'-R(*GP*CP*UP*AP*CP*UP*AP*A)-3')
  • RNA (5'-R(P*AP*GP*UP*AP*GP*UP*GP*UP*GP*C)-3')
  • RNA (5'-R(P*UP*UP*AP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
  • RNA-directed RNA polymerase L
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
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
La Crosse orthobunyavirus
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.961 Å
AuthorsCusack, S. / Gerlach, P. / Reguera, J.
CitationJournal: Nat Commun / Year: 2020
Title: Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes.
Authors: Benoît Arragain / Grégory Effantin / Piotr Gerlach / Juan Reguera / Guy Schoehn / Stephen Cusack / Hélène Malet /
Abstract: Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening pathogens against which no effective treatment is currently available. Replication and ...Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening pathogens against which no effective treatment is currently available. Replication and transcription of the RNA genome constitute essential processes performed by the virally encoded multi-domain RNA-dependent RNA polymerase. Here, we describe the complete high-resolution cryo-EM structure of La Crosse virus polymerase. It reveals the presence of key protruding C-terminal domains, notably the cap-binding domain, which undergoes large movements related to its role in transcription initiation, and a zinc-binding domain that displays a fold not previously observed. We capture the polymerase structure at pre-initiation and elongation states, uncovering the coordinated movement of the priming loop, mid-thumb ring linker and lid domain required for the establishment of a ten-base-pair template-product RNA duplex before strand separation into respective exit tunnels. These structural details and the observed dynamics of key functional elements will be instrumental for structure-based development of polymerase inhibitors.
History
DepositionMay 28, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 29, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 24, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: atom_type / chem_comp_atom ...atom_type / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z ..._atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
BBB: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*GP*UP*GP*C)-3')
CCC: RNA (5'-R(P*UP*UP*AP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
DDD: RNA (5'-R(*GP*CP*UP*AP*CP*UP*AP*A)-3')
EEE: RNA-directed RNA polymerase L
HHH: RNA (5'-R(P*UP*UP*AP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
PPP: RNA-directed RNA polymerase L
UUU: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*GP*UP*GP*C)-3')
XXX: RNA (5'-R(*GP*CP*UP*AP*CP*UP*AP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)553,77110
Polymers553,6408
Non-polymers1312
Water00
1
BBB: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*GP*UP*GP*C)-3')
CCC: RNA (5'-R(P*UP*UP*AP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
DDD: RNA (5'-R(*GP*CP*UP*AP*CP*UP*AP*A)-3')
EEE: RNA-directed RNA polymerase L
hetero molecules


Theoretical massNumber of molelcules
Total (without water)276,8855
Polymers276,8204
Non-polymers651
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7880 Å2
ΔGint-51 kcal/mol
Surface area89630 Å2
MethodPISA
2
HHH: RNA (5'-R(P*UP*UP*AP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
PPP: RNA-directed RNA polymerase L
UUU: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*GP*UP*GP*C)-3')
XXX: RNA (5'-R(*GP*CP*UP*AP*CP*UP*AP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)276,8855
Polymers276,8204
Non-polymers651
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7820 Å2
ΔGint-49 kcal/mol
Surface area89190 Å2
MethodPISA
Unit cell
Length a, b, c (Å)371.187, 145.320, 234.193
Angle α, β, γ (deg.)90.000, 116.333, 90.000
Int Tables number5
Space group name H-MC121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11Chains CCC HHH
22Chains EEE PPP
/ NCS ensembles :
ID
2
1

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Components

#1: RNA chain RNA (5'-R(P*AP*GP*UP*AP*GP*UP*GP*UP*GP*C)-3')


Mass: 3217.956 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: vRNA 5' end (1-10) / Source: (synth.) La Crosse orthobunyavirus
#2: RNA chain RNA (5'-R(P*UP*UP*AP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')


Mass: 5044.023 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: vRNA 3' end (1-16) / Source: (synth.) La Crosse orthobunyavirus
#3: RNA chain RNA (5'-R(*GP*CP*UP*AP*CP*UP*AP*A)-3')


Mass: 2510.561 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: vRNA 5' end (9-16) / Source: (synth.) La Crosse orthobunyavirus
#4: Protein RNA-directed RNA polymerase L / Protein L / Large structural protein / Replicase / Transcriptase


Mass: 266047.469 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: N-terminal His-tag / Source: (gene. exp.) Bunyavirus La Crosse / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: A5HC98, RNA-directed RNA polymerase, Hydrolases; Acting on ester bonds
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: 5.42 Å3/Da / Density % sol: 77.33 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8
Details: LACV-L in complex with pre-annealed 3 prime (1-16) and 5 prime (9-16) vRNA was concentrated to 5 mg/ml. The 5 prime (1-10) vRNA end was later soaked into crystals in 1 to 2 molar ratio. ...Details: LACV-L in complex with pre-annealed 3 prime (1-16) and 5 prime (9-16) vRNA was concentrated to 5 mg/ml. The 5 prime (1-10) vRNA end was later soaked into crystals in 1 to 2 molar ratio. Mother liquor was 100 mM Tris pH 8.0, 100 mM NaCl, and 8% PEG 4000. Crystals were soaked in a stepwise manner with increasing concentration of the glycerol cryo-protectant, reaching 30%

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 0.9724 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Nov 26, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9724 Å / Relative weight: 1
ReflectionResolution: 3.96→209.891 Å / Num. obs: 59211 / % possible obs: 60.8 % / Redundancy: 11.6 % / CC1/2: 0.995 / Rrim(I) all: 0.163 / Net I/σ(I): 6.5
Reflection shellResolution: 3.96→4.35 Å / Num. unique obs: 2961 / CC1/2: 0.47 / Rrim(I) all: 1.285

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
XDSdata reduction
STARANISOdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5AMR

5amr


Resolution: 3.961→209.891 Å / Cor.coef. Fo:Fc: 0.855 / Cor.coef. Fo:Fc free: 0.828 / SU B: 62.711 / SU ML: 0.824 / Cross valid method: FREE R-VALUE / ESU R Free: 1.092
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2988 1514 2.557 %
Rwork0.2638 57697 -
all0.265 --
obs-59211 60.786 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 189.756 Å2
Baniso -1Baniso -2Baniso -3
1-2.243 Å20 Å2-1.674 Å2
2--1.232 Å20 Å2
3----1.22 Å2
Refinement stepCycle: LAST / Resolution: 3.961→209.891 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms33204 1434 2 0 34640
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0020.01335476
X-RAY DIFFRACTIONr_bond_other_d0.0010.01732445
X-RAY DIFFRACTIONr_ext_dist_refined_d0.2220.012315
X-RAY DIFFRACTIONr_angle_refined_deg1.1561.62348135
X-RAY DIFFRACTIONr_angle_other_deg1.0411.60775587
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.01754046
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.95423.0121776
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.299156441
X-RAY DIFFRACTIONr_dihedral_angle_4_deg11.11515180
X-RAY DIFFRACTIONr_chiral_restr0.0380.24769
X-RAY DIFFRACTIONr_gen_planes_refined0.0020.0237770
X-RAY DIFFRACTIONr_gen_planes_other0.0010.027382
X-RAY DIFFRACTIONr_nbd_refined0.170.26345
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1720.229879
X-RAY DIFFRACTIONr_nbtor_refined0.1590.215876
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0740.216211
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.150.2452
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.0660.22
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1350.213
X-RAY DIFFRACTIONr_nbd_other0.1820.256
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.3480.21
X-RAY DIFFRACTIONr_mcbond_it6.30720.3216247
X-RAY DIFFRACTIONr_mcbond_other6.30420.3216246
X-RAY DIFFRACTIONr_mcangle_it11.06430.45620272
X-RAY DIFFRACTIONr_mcangle_other11.06430.45620273
X-RAY DIFFRACTIONr_scbond_it4.78120.6519229
X-RAY DIFFRACTIONr_scbond_other4.78120.6519230
X-RAY DIFFRACTIONr_scangle_it8.9830.78927863
X-RAY DIFFRACTIONr_scangle_other8.9830.78927864
X-RAY DIFFRACTIONr_lrange_it17.253238.3340160
X-RAY DIFFRACTIONr_lrange_other17.253238.32740161
X-RAY DIFFRACTIONr_ncsr_local_group_10.0050.051341
X-RAY DIFFRACTIONr_ncsr_local_group_20.0390.0566283
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc freeFsc work% reflection obs (%)WRfactor Rwork
3.961-4.0630.3740.3231750.32471860.7940.7582.4910.328
4.063-4.1750.387140.34310.30369480.6970.7686.40470.3
4.175-4.2960.273400.32513540.32468080.7290.72620.47590.324
4.296-4.4280.34790.31122590.31266050.7730.75835.39740.305
4.428-4.5730.367720.28927880.29163450.7790.78845.07490.28
4.573-4.7330.285720.26532830.26662030.7910.82154.08670.251
4.733-4.9120.29770.26336810.26460000.8350.83762.63330.245
4.912-5.1120.2931110.25239060.25357520.840.86469.83660.23
5.112-5.340.2781060.24639930.24755060.8640.86974.44610.223
5.34-5.60.2761060.24340060.24453000.8670.87477.58490.22
5.6-5.9030.2871080.23839860.23950140.840.8781.65140.215
5.903-6.2610.2811150.2440270.24147750.8710.8786.74350.215
6.261-6.6920.2761040.24540430.24644670.8570.86292.83640.22
6.692-7.2280.3011030.24440120.24641750.8580.86998.56290.225
7.228-7.9170.2681010.2337460.23138560.8960.89399.76660.219
7.917-8.850.286750.21133910.21334800.8940.91899.59770.215
8.85-10.2160.232990.20329590.20431100.9250.92898.3280.215
10.216-12.5040.269600.23325570.23426260.8920.90899.65730.26
12.504-17.6530.307360.32119880.3220400.8490.84199.21570.361
17.653-209.8910.554320.56911120.56911830.6960.69396.70330.761

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