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- EMDB-23345: Cryo-EM structure of Mycobacterium smegmatis Lhr helicase C-termi... -

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Basic information

Entry
Database: EMDB / ID: EMD-23345
TitleCryo-EM structure of Mycobacterium smegmatis Lhr helicase C-terminal domain
Map dataCryo-EM structure of Mycobacterium smegmatis Lhr helicase C-terminal domain
Sample
  • Complex: Tetramer structure of ATP-dependent DNA helicase Lhr C-terminal domain
    • Protein or peptide: ATP-dependent DNA helicase
Keywordshelicase / winged helix / HYDROLASE
Function / homology
Function and homology information


hydrolase activity, acting on acid anhydrides, in phosphorus-containing anhydrides / RNA helicase activity / nucleic acid binding / RNA helicase / ATP binding
Similarity search - Function
DEAD/H associated / DEAD/H associated / DNA glycosylase AlkZ-like / DNA glycosylase AlkZ-like / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. ...DEAD/H associated / DEAD/H associated / DNA glycosylase AlkZ-like / DNA glycosylase AlkZ-like / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ATP-dependent DNA helicase
Similarity search - Component
Biological speciesMycolicibacterium smegmatis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWang J / Warren GM
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)P30CA008748 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI64693 United States
CitationJournal: Nucleic Acids Res / Year: 2021
Title: Oligomeric quaternary structure of Escherichia coli and Mycobacterium smegmatis Lhr helicases is nucleated by a novel C-terminal domain composed of five winged-helix modules.
Authors: Garrett M Warren / Juncheng Wang / Dinshaw J Patel / Stewart Shuman /
Abstract: Mycobacterium smegmatis Lhr (MsmLhr; 1507-aa) is the founder of a novel clade of bacterial helicases. MsmLhr consists of an N-terminal helicase domain (aa 1-856) with a distinctive tertiary structure ...Mycobacterium smegmatis Lhr (MsmLhr; 1507-aa) is the founder of a novel clade of bacterial helicases. MsmLhr consists of an N-terminal helicase domain (aa 1-856) with a distinctive tertiary structure (Lhr-Core) and a C-terminal domain (Lhr-CTD) of unknown structure. Here, we report that Escherichia coli Lhr (EcoLhr; 1538-aa) is an ATPase, translocase and ATP-dependent helicase. Like MsmLhr, EcoLhr translocates 3' to 5' on ssDNA and unwinds secondary structures en route, with RNA:DNA hybrid being preferred versus DNA:DNA duplex. The ATPase and translocase activities of EcoLhr inhere to its 877-aa Core domain. Full-length EcoLhr and MsmLhr have homo-oligomeric quaternary structures in solution, whereas their respective Core domains are monomers. The MsmLhr CTD per se is a homo-oligomer in solution. We employed cryo-EM to solve the structure of the CTD of full-length MsmLhr. The CTD protomer is composed of a series of five winged-helix (WH) modules and a β-barrel module. The CTD adopts a unique homo-tetrameric quaternary structure. A Lhr-CTD subdomain, comprising three tandem WH modules and the β-barrel, is structurally homologous to AlkZ, a bacterial DNA glycosylase that recognizes and excises inter-strand DNA crosslinks. This homology is noteworthy given that Lhr is induced in mycobacteria exposed to the inter-strand crosslinker mitomycin C.
History
DepositionJan 25, 2021-
Header (metadata) releaseApr 7, 2021-
Map releaseApr 7, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7lhl
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23345.png

Downloads & links

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Map

FileDownload / File: emd_23345.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of Mycobacterium smegmatis Lhr helicase C-terminal domain
Voxel sizeX=Y=Z: 1.064 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.067690015 - 0.1017085
Average (Standard dev.)-0.000009072544 (±0.0036283494)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 272.384 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0641.0641.064
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z272.384272.384272.384
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ160160160
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0680.102-0.000

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Supplemental data

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Sample components

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Entire : Tetramer structure of ATP-dependent DNA helicase Lhr C-terminal domain

EntireName: Tetramer structure of ATP-dependent DNA helicase Lhr C-terminal domain
Components
  • Complex: Tetramer structure of ATP-dependent DNA helicase Lhr C-terminal domain
    • Protein or peptide: ATP-dependent DNA helicase

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Supramolecule #1: Tetramer structure of ATP-dependent DNA helicase Lhr C-terminal domain

SupramoleculeName: Tetramer structure of ATP-dependent DNA helicase Lhr C-terminal domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Molecular weightTheoretical: 260 kDa/nm

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Macromolecule #1: ATP-dependent DNA helicase

MacromoleculeName: ATP-dependent DNA helicase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: RNA helicase
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Molecular weightTheoretical: 162.009562 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SMTTNGADPL GRFSALTREW FTTAFAAPTP AQADAWSAIS EGNNTLVIAP TGSGKTLAAF LWAIDRLADP AREPSQGTQV LYVSPLKAL AVDVERNLRT PLTGITRVAE RHGLPAPSIT VGVRSGDTPP NQRRAMIANP PDVLITTPES LFLMLTSAAR E TLTSVRTV ...String:
SMTTNGADPL GRFSALTREW FTTAFAAPTP AQADAWSAIS EGNNTLVIAP TGSGKTLAAF LWAIDRLADP AREPSQGTQV LYVSPLKAL AVDVERNLRT PLTGITRVAE RHGLPAPSIT VGVRSGDTPP NQRRAMIANP PDVLITTPES LFLMLTSAAR E TLTSVRTV IVDEVHAVAA TKRGAHLALS LERLDQLLDT PAQRIGLSAT VRPPEEVARF LSGQAPTTIV CPPAAKTFDL SV QVPVPDM ANLDNNSIWP DVEERIVDLV EAHNSSIVFA NSRRLAERLT SRLNEIHAER SGIELPAGPN PEVGGGAPAH LMG SGQANG APPLLARAHH GSVSKEQRAQ VEDDLKSGRL RAVVATSSLE LGIDMGAVDL VIQVEAPPSV ASGLQRVGRA GHQV GEISQ GVLFPKHRTD LIGCAVTVQR MQTGDIETLR VPANPLDVLA QHTVAVAALE PVDADAWFDA VRRSAPFATL PRSAF EATL DLLSGKYPST EFAELRPRLV YDRDTGTLTA RPGAQRLAVT SGGAIPDRGM FTVYLASETE KPSRVGELDE EMVYES RPG DVISLGATSW RITEITHDRV LVIPAPGQPA RLPFWRGDSV GRPAELGAAV GAFTGELASL DRKAFDKRCQ KMGFAGY AT DNLHQLLREQ REATGVVPSD TTFVVERFRD ELGDWRVILH SPYGLRVHGP LALAVGRRLR ERYGIDEKPT ASDDGIIV R LPDSGDTPPG ADLFVFDADE IEPIVTAEVG GSALFASRFR ECAARALLLP RRHPGKRSPL WHQRQRAAQL LDIARKYPD FPIVLEAVRE CLQDVYDVPA LIELMHKIAQ RRLRIVEVET ATPSPFAASL LFGYVGAFMY EGDSPLAERR AAALALDTVL LSELLGRVE LRELLDPAVV ASTSAQLQHL TPERAARDAE GVADLLRLLG PLTEADIAQR CTADNIGAWL DGLHAAKRAL P VTYAGQTW WAAVEDIGLL RDGIGVPVPV GVPAAFTESA SDPLGDLIGR YARTRGPFTT EQTAARFGLG VRVASDVLSR MA VDGRLIR GEFAADLSGE QWCDAQVLKI LRRRSLAALR AQVEPVSTDA YARFLPSWQH VGSTNTTGVD GLATVIEQLA GVP IPASAV ESLVFPQRVR DYQPAMLDEL LASGEVMWSG AGQIGNGDGW VAFHLADTAP LTLTHGAEIE FTDTHRVILE TLGH GGAYF FRQLTDGTVE GTAGQELKQA LWELIWAGWV TGDTFAPVRA VLSGPRRSGA PAHRQRQRPP RLSRYSVAHA QTRGT DPTV SGRWSALPAA EPDSTVRAHF QAELLLGRHG VLTKGAVGAE GVPGGFATLY KVLSAFEDAG RCQRGYFVES LGGAQF AVA STVDRLRSYL DNVDPERPEY HAVVLAATDP ANPYGAALGW PTDSEAHRPG RKAGALVALV DGRLVWFLER GGRSLLS FG ADADAQRAAA GALTDLVSAG RIPSLLVERI NGVAVLDPDV DAERAVVQDA LLGAGLSRTP RGLRLR

UniProtKB: ATP-dependent DNA helicase

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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

Concentration0.3 mg/mL
BufferpH: 7.5
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 47262 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 53.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 101323

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