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- EMDB-7941: Fako virus empty particles aligned to the best decoy map (asymmet... -

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Basic information

Entry
Database: EMDB / ID: EMD-7941
TitleFako virus empty particles aligned to the best decoy map (asymmetric reconstruction) showing the locations of the polymerase complexes
Map dataAsymmetric reconstruction of Fako virus empty particles aligned to the best decoy map, showing the locations of the polymerase complexes
Sample
  • Virus: Fako virus
    • Protein or peptide: major capsid protein
    • Protein or peptide: RNA-dependent RNA polymerase
    • Protein or peptide: turret
    • Protein or peptide: clamp
    • Protein or peptide: accessory NTPase
Biological speciesFako virus
Methodsingle particle reconstruction / cryo EM
AuthorsKaelber JT / Jiang W / Weaver SC / Auguste AJ / Chiu W
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesP41GM103832 United States
Welch FoundationQ1242 United States
CitationJournal: Structure / Year: 2020
Title: Arrangement of the Polymerase Complexes inside a Nine-Segmented dsRNA Virus.
Authors: Jason T Kaelber / Wen Jiang / Scott C Weaver / Albert J Auguste / Wah Chiu /
Abstract: Members of the family Reoviridae package several copies of the viral polymerase complex into their capsid to carry out replication and transcription within viral particles. Classical single-particle ...Members of the family Reoviridae package several copies of the viral polymerase complex into their capsid to carry out replication and transcription within viral particles. Classical single-particle reconstruction encounters difficulties resolving structures such as the intraparticle polymerase complex because refinement can converge to an incorrect map and because the map could depict a nonrepresentative subset of particles or an average of heterogeneous particles. Using the nine-segmented Fako virus, we tested hypotheses for the arrangement and number of polymerase complexes within the virion by measuring how well each hypothesis describes the set of cryoelectron microscopy images of individual viral particles. We find that the polymerase complex in Fako virus binds at ten possible sites despite having only nine genome segments. A single asymmetric configuration describes the arrangement of these complexes in both virions and genome-free capsids. Similarities between the arrangements of Reoviridae with 9, 10, and 11 segments indicate the generalizability of this architecture.
History
DepositionMay 26, 2018-
Header (metadata) releaseJan 16, 2019-
Map releaseJan 16, 2019-
UpdateAug 5, 2020-
Current statusAug 5, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 30
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 30
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7941.png

Downloads & links

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Map

FileDownload / File: emd_7941.map.gz / Format: CCP4 / Size: 253.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAsymmetric reconstruction of Fako virus empty particles aligned to the best decoy map, showing the locations of the polymerase complexes
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.14 Å/pix.
x 405 pix.
= 867.51 Å		2.14 Å/pix.
x 405 pix.
= 867.51 Å		2.14 Å/pix.
x 405 pix.
= 867.51 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.142 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 30 / Movie #1: 30
Minimum - Maximum-66.40891 - 99.88296
Average (Standard dev.)0.61569375 (±7.19561)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-202-202-202
Dimensions405405405
Spacing405405405
CellA=B=C: 867.51 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.1422.1422.142
M x/y/z405405405
origin x/y/z0.0000.0000.000
length x/y/z867.510867.510867.510
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ352352352
MAP C/R/S123
start NC/NR/NS-202-202-202
NC/NR/NS405405405
D min/max/mean-66.40999.8830.616

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Supplemental data

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Sample components

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Entire : Fako virus

EntireName: Fako virus
Components
  • Virus: Fako virus
    • Protein or peptide: major capsid protein
    • Protein or peptide: RNA-dependent RNA polymerase
    • Protein or peptide: turret
    • Protein or peptide: clamp
    • Protein or peptide: accessory NTPase

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Supramolecule #1: Fako virus

SupramoleculeName: Fako virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 1567404 / Sci species name: Fako virus / Sci species strain: CSW77 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Culicinae (mosquito)
Molecular weightTheoretical: 43 MDa
Virus shellShell ID: 1 / T number (triangulation number): 2

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Macromolecule #1: major capsid protein

MacromoleculeName: major capsid protein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Fako virus / Strain: CSW77
SequenceString: MRPIRMYKNN QERTNLKHQE INEEQQNEQT TSNQGFTRSD NSGKINIERI SSSRNQITDG KTVSSYSKI ETNRSSQDSV QHGGSSITYT SDTTGNPRIT NARTNNDETH ATGPIEDLNS T SHGREPEI ESFADRAELA MMIQGMTVGA LTVQPMRSIR STFANLANVL ...String:
MRPIRMYKNN QERTNLKHQE INEEQQNEQT TSNQGFTRSD NSGKINIERI SSSRNQITDG KTVSSYSKI ETNRSSQDSV QHGGSSITYT SDTTGNPRIT NARTNNDETH ATGPIEDLNS T SHGREPEI ESFADRAELA MMIQGMTVGA LTVQPMRSIR STFANLANVL IFHDVFTTED KP SAFIEYH SDEMIVNMPK QTYNPIDNLA KILYLPSLEK FKYGTGIVQL NYSPHISKLY QNT NNIINT ITDGITYANR TEFFIRVMVL MMMDRKILTM EFYDVDTSAI SNTAILPTIP TTTG VSPLL RIDTRTEPIW YNDAIKTLIT NLTIQYGKIK TVLDANAVKR YSVVGYPIDQ YRAYL YNHN LLEYLGKKVK REDIMSLIKA LSYEFDLITI SDLEYQNIPK WFSDNDLSRF IFSICM FPD IVRQFHALNI DYFSQANVFT VKSENAIVKM LNSNQNMEPT IINWFLFRIC AIDKTVI DD YFSLEMTPII MRPKLYDFDM KRGEPVSLLY ILELILFSIM FPNVTQHMLG QIQARILY I SMYAFRQEYL KFITKFGFYY KIVNGRKEYI QVTNQNERMT ENNDVLTGNL YPSLFTDDP TLSAIAPTLA KIARLMKPTT SLTPDDRAIA AKFPRFKDSA HLNPYSSLNI GGRTQHSVTY TRMYDAIEE MFNLILRAFA SSFAQRPRAG VTQLKSLLTQ LADPLCLALD GHVYHLYNVM A NMMQNFIP NTDGQFHSFR ACSYAVKDGG NIYRVVQNGD ELNESLLIDT AIVWGLLGNT DS SYGNAIG ATGTANVPTK VQPVIPTPDN FITPTIHLKT SIDAICSVEG ILLLILSRQT TIP GYEDEL NKLRTGISQP KVTERQYRRA RESIKNMLGS GDYNVAPLHF LLHTEHRSTK LSKP LIRRV LDNVVQPYVA NLDPAEFENT PQLIENSNMT RLQIALKMLT GDMDDIVKGL ILHKR ACAK FDVYETLTIP TDVKTIVLTM QHISTQTQNN MVYYVFLIDG VKILAEDIKN VNFQID ITG IWPEYVITLL LRAINNGFNT YVSMPNILYK PTITADVRQF MNTTKAETLL ISNKSIV HE IMFFDNALQP KMSSDTLALS EAVYRTIWNS SIITQRISAR GLMNLEDARP PEAKISHQ S ELDMGKIDET SGEPIYTSGL QKMQSSKVSM ANVVLSAGSD VIRQAAIKYN VVRTQEIIL FE

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Macromolecule #2: RNA-dependent RNA polymerase

MacromoleculeName: RNA-dependent RNA polymerase / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Fako virus / Strain: CSW77
SequenceString: MATEPNEINL RMTQTLMKQI EDTTFKIRRK HDDMYTYLID DTAAIEMYPD TETPFRMKSS LHELYIQVK CQIPKSIIRP PLYNNDMYPL NNNNTNFLED RPFNYLCNFD WYEFLSRTKD E LGMHYNMI RDLIAMSSQT RYSNIWSNIS GIIMYLRQYQ RGFALRAILA ...String:
MATEPNEINL RMTQTLMKQI EDTTFKIRRK HDDMYTYLID DTAAIEMYPD TETPFRMKSS LHELYIQVK CQIPKSIIRP PLYNNDMYPL NNNNTNFLED RPFNYLCNFD WYEFLSRTKD E LGMHYNMI RDLIAMSSQT RYSNIWSNIS GIIMYLRQYQ RGFALRAILA RLLKQWNNFP FF DTWDGFR DVLPNTSTAW PLLFYAFMSV TFDYICENIS EGEAIVCFQN HLENAQVSYQ DTK LEKKNN YTLWLKYECH NLRVALTPRY DYTGTVSGNF LKDTTEYIIE NEFENNEIAM RNNL LPSFY AILQQMRQKL KTVEDIIRLL TICYSARDDR TYYGTLMELA ISKAIKPQVA GSIVP APIP TSWLQKDPKM VLSARYPSTS FLSQMQEFYR RYYPALQKEI DVHALSASFI NFLSTA SAG VGIELPEEIV NMVQDKRLLY LIKKGSGKRV LQEALLVEKY NDLDPIINDF VRLIKIV VR KQIERRQRGI AGIPNNVLKI NQVTYEANKP FSKIARAPSH GKQSGNASDI HDLLFYTT Q EDVSHIETNG KRQMRGIVIS SADVKGMDTH IQINAAMNQH LGAIEILDGI QYDVGPFRQ TNAIIQDIQG NVYEKSLNGG QQAIAFGLAN FSQTTGINSK YFGQIPNQEG TFPSGLITTS NHHTQMLTL LIETALTTFT KEFGKSMAIS HLMILGDDVS LMLHGNDKDI NFFMKYLVEK F SQLGLILE RDESRNFGVF LQQHVINGRF NGFSNRIAIF TSEDYKTRKS VRESCTEYNA LI DDVIFRT YNVRKLLQFQ RIHQFVVLSK YVFRIQNYKY ESLRAKLAKR MNVFEYELKI KDN DKTDVH NRNALRFIGI QIPYTYFQYS GGGEIPPESF QKKDGSFTYE YSIYSPRGKW LRKF LYDIS HDARDVKFQI DHEMMKLYNL DICDFLLQYN VLDIQEEIRS TIIDRELVSK LAMNL ESLE HSNARMISRR ASESLRSMGI RLPANGVYGY QINERLVKVL QNIQQSDYEV KMVGDT LFT AIMEKFEHHK VRMEKGDKLH NFTLDFSNKD DKITINRKML ALHNISISKN MMPYSDA WM LYTCLDNTYN TSSDLATALA HSQGHFKSFQ YDRDMFAESV KIASKHGIGS LPMELFFE A SNIRDTAQLK WIEAIKYYVQ FKDYLYPYSL NPRRLFFIPE QVSSVSNILN QENIPADIN QKALLFRRAY AYVLSHPFCI SGAKAIFVED RIS

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Macromolecule #3: turret

MacromoleculeName: turret / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Fako virus / Strain: CSW77
SequenceString: MIDLRLEEDI LTATLPEFLS TRPKYRYAYT NTKQQDIRFQ GPMRHVRLTH LYKQTKLWNL QYIERELAI SEIDDALDEF IQTFSLPYVI EQGTYKYNML LGMHAHNVNY QDDVSELIAN N PQLLNYLD DNPFSAIFEL VNVDLQIYQY GQNIFNNEAE HTILFLKDNT ...String:
MIDLRLEEDI LTATLPEFLS TRPKYRYAYT NTKQQDIRFQ GPMRHVRLTH LYKQTKLWNL QYIERELAI SEIDDALDEF IQTFSLPYVI EQGTYKYNML LGMHAHNVNY QDDVSELIAN N PQLLNYLD DNPFSAIFEL VNVDLQIYQY GQNIFNNEAE HTILFLKDNT NYGVIQALQK HP FSATHIN WHLHKHIFVF HSREQLLNKL LSAGLEDSQL YQRQKTYSTK RGDRPTERMV TYI EDDHIR RIQAVFPLLL DNIFDVKLHK DSSMTWLKSY ADMIYDSVKN SNSTITPEIR KLYL RMYNQ YMRIFLPIEQ YMLYDNTCWP FSEKITLKIN VRLISSRENQ PVLWKTPIDT ENLIS IVQP DEPINKLNFT AIPSTMIRLN DNITMYRAVK DMFSAIEYLP DAIENIPTLT MKEQAL SRY ISPDSEAQNF FNNQPPYLNS IMNVNRQVFE AVKRGNIQVS TGSMEHLCLC MHVKSGL IV GRTVLIDDKV VLRRNFNAST AKMITCYVKA FAQLYGEGSL INPGLRMVFF GVETEPAI D ILKLFYGDKS LYIQGFGDRG IGRDKFRTKI EDALTLRIGC DILISDIDQA DYEDPNEEK FDDITDFVCY VTELVISNAT VGLVKISMPT YYIMNKISST LNNKFSNVAI NIVKLSTQKP YTYEAYIML SHGSTLTNKG YLRNPVCDVY LEKISLQPMD LKIISTISNE INYDKPTLYR F VVDKNDVT DVSIAMHILS IHCSTITTRS VMVRSDNTGA FVTMSGIKDM KRVAIMNRMT DG TSANSYM HEQNGKLYLQ KVPYLEDLIS AFPNGFGSTY QNDYDSSMSV INVNALIRQV VYR VISKSI PVALLESLSR IRIIGGRDLG EMNAVYKLYK TPIEVYDAVG ITREYPHVQI SYRA QRYSF TESIPNHTLL LANYVIMNDV DGAPISSLEQ INTIKKIISK ISLGSIAYIQ VYTDI VARN INVMTKNDSF LISANADKTV FKVQVSGYKA VEMCNYEQLL QLVSDNTGVN IIKLTY QDV LESCVLSSGI LGDTGSWLLD LVLASTYIIE IRG

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Macromolecule #4: clamp

MacromoleculeName: clamp / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Fako virus / Strain: CSW77
SequenceString: MTLTYWDKEK RMTLKQMIQQ VAINEQENEL THYVFTTPLS MPTFGKPMLG YVPLNEVATS KFFSNVNDF DRDNQLAMAH FPDTTITQAY NLTNSIKPGD TSLPDAEVAA LKWFWKFFTS I NLVRQPPM DNVMYWACQF LSSGTSFLPL ERDVEIVFSG FKGSHICMFS ...String:
MTLTYWDKEK RMTLKQMIQQ VAINEQENEL THYVFTTPLS MPTFGKPMLG YVPLNEVATS KFFSNVNDF DRDNQLAMAH FPDTTITQAY NLTNSIKPGD TSLPDAEVAA LKWFWKFFTS I NLVRQPPM DNVMYWACQF LSSGTSFLPL ERDVEIVFSG FKGSHICMFS NLRQMNLSPI LC PYYDLIT NFKTTTEIRA YVDAHEELKS LLTYLCLCTI VGLCDTFTET RNMDTGEYVW KVR DVVSRN HTPAQNVEKF CYTIQNAKYM IQLVHVLLFP LTDNKYADLP NYVAVITQGA INQS RSHNV INTTDESNSN TTSDTAASTS GIVSGDTGTV ASLYPDEFKY VQS

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Macromolecule #5: accessory NTPase

MacromoleculeName: accessory NTPase / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Fako virus / Strain: CSW77
SequenceString: MFAIPFTASQ VYSHLGLYQQ PIEKLDPLTK VQYESAEKLR DIITQLSRRS GGISLRKNLI RDIAFGNKP KLNIPSLTHQ VRHNREFKHG RGQFILVDSN GSDYYDGIFD DMVLILRSNF V LPNVPGII SEQLRLIVID NDFYLKHIPS EIAILRDVLS EHMVKVSIDG ...String:
MFAIPFTASQ VYSHLGLYQQ PIEKLDPLTK VQYESAEKLR DIITQLSRRS GGISLRKNLI RDIAFGNKP KLNIPSLTHQ VRHNREFKHG RGQFILVDSN GSDYYDGIFD DMVLILRSNF V LPNVPGII SEQLRLIVID NDFYLKHIPS EIAILRDVLS EHMVKVSIDG VSLDDPYISK FF RKPFILY DHTKFRKVDL TKVVLINKTG KSKAQLIDKY KLTDDAVFIS YEVFDIMLQP VKS RDGDIE QFVRMRGDIE TWKMKIGTTF ESNLMELFIH GVPVMNSTSQ LQLDFQISDI RSAN VFDEQ IVCAAKLNGL REQCLKESSL IRVNIIGQKG SGKSMLMRLI NERGIPGISR KIICI DSDA YGKWKTQTQY LDDKFSALKL INVDNLHEIS QDDNIISYYE QFIIDQLLAH NITISE HTN SIRFIKQFKV DTLKDIGRKF KELYLADFKE QIHFYEYLCG NIPEPSSTLL ITFLHAT VE TSAAPGTNMN FSLNTILYPL QSILNRKRGA LVNFILNRIY DEMGTEAFTR LRPCDVWK

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.8
Component:
ConcentrationFormulaName
10.0 mMC4H12NO3CLTris-HCl
1.0 mMEDTA
100.0 mMNaClsodium chloride
GridPretreatment - Type: PLASMA CLEANING / Details: unspecified
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL 3200FSC
Specialist opticsEnergy filter - Name: In-column Omega Filter
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Detector mode: INTEGRATING / Number real images: 2400 / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 4.7 mm
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 9772
CTF correctionSoftware - Name: jspr
Startup modelType of model: EMDB MAP
EMDB ID:

6001

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution method: OTHER / Software - Name: jspr
Details: The reconstruction method used does not preserve the independence of half-sets. As such, the Fourier shell correlation between half-sets is a biased measure and resolution is not calculated.
Number images used: 4886
Initial angle assignmentType: OTHER / Software - Name: jspr
Final angle assignmentType: PROJECTION MATCHING / Software - Name: jspr

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