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- EMDB-41097: cryoEM structure of Smc5/6 5mer -

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Basic information

Entry
Database: EMDB / ID: EMD-41097
TitlecryoEM structure of Smc5/6 5mer
Map datasharppen map, zflip
Sample
  • Complex: Smc5/6 5mer
    • Protein or peptide: Structural maintenance of chromosomes protein 6
    • Protein or peptide: Non-structural maintenance of chromosome element 5
    • Protein or peptide: DNA repair protein KRE29
KeywordsSmc / Nse / Condensin / Cohesin / DNA binding / DNA damage / DNA repair / DNA BINDING PROTEIN
Function / homology
Function and homology information


Smc5-Smc6 complex / resolution of DNA recombination intermediates / DNA double-strand break attachment to nuclear envelope / chromosome separation / SUMOylation of DNA damage response and repair proteins / chromatin looping / regulation of telomere maintenance / protein sumoylation / double-strand break repair via homologous recombination / single-stranded DNA binding ...Smc5-Smc6 complex / resolution of DNA recombination intermediates / DNA double-strand break attachment to nuclear envelope / chromosome separation / SUMOylation of DNA damage response and repair proteins / chromatin looping / regulation of telomere maintenance / protein sumoylation / double-strand break repair via homologous recombination / single-stranded DNA binding / site of double-strand break / damaged DNA binding / chromosome, telomeric region / DNA repair / ATP hydrolysis activity / mitochondrion / ATP binding / nucleus / cytoplasm
Similarity search - Function
DNA repair protein Nse5/Nse6 / DNA repair proteins Nse5 and Nse6 / Rad50/SbcC-type AAA domain / AAA domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA repair protein KRE29 / Non-structural maintenance of chromosome element 5 / Structural maintenance of chromosomes protein 6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae W303 (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsYu Y / Patel DJ
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)GM145260 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Molecular basis for Nse5-6 mediated regulation of Smc5/6 functions.
Authors: Shibai Li / You Yu / Jian Zheng / Victoria Miller-Browne / Zheng Ser / Huihui Kuang / Dinshaw J Patel / Xiaolan Zhao /
Abstract: The Smc5/6 complex (Smc5/6) is important for genome replication and repair in eukaryotes. Its cellular functions are closely linked to the ATPase activity of the Smc5 and Smc6 subunits. This activity ...The Smc5/6 complex (Smc5/6) is important for genome replication and repair in eukaryotes. Its cellular functions are closely linked to the ATPase activity of the Smc5 and Smc6 subunits. This activity requires the dimerization of the motor domains of the two SMC subunits and is regulated by the six non-SMC subunits (Nse1 to Nse6). Among the NSEs, Nse5 and Nse6 form a stable subcomplex (Nse5-6) that dampens the ATPase activity of the complex. However, the underlying mechanisms and biological significance of this regulation remain unclear. Here, we address these issues using structural and functional studies. We determined cryo-EM structures of the yeast Smc5/6 derived from complexes consisting of either all eight subunits or a subset of five subunits. Both structures reveal that Nse5-6 associates with Smc6's motor domain and the adjacent coiled-coil segment, termed the neck region. Our structural analyses reveal that this binding is compatible with motor domain dimerization but results in dislodging the Nse4 subunit from the Smc6 neck. As the Nse4-Smc6 neck interaction favors motor domain engagement and thus ATPase activity, Nse6's competition with Nse4 can explain how Nse5-6 disfavors ATPase activity. Such regulation could in principle differentially affect Smc5/6-mediated processes depending on their needs of the complex's ATPase activity. Indeed, mutagenesis data in cells provide evidence that the Nse6-Smc6 neck interaction is important for the resolution of DNA repair intermediates but not for replication termination. Our results thus provide a molecular basis for how Nse5-6 modulates the ATPase activity and cellular functions of Smc5/6.
History
DepositionJun 22, 2023-
Header (metadata) releaseNov 15, 2023-
Map releaseNov 15, 2023-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41097.png

Downloads & links

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Map

FileDownload / File: emd_41097.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharppen map, zflip
Voxel sizeX=Y=Z: 1.064 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-3.0059516 - 4.829931
Average (Standard dev.)0.0001336502 (±0.062087663)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 340.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A, zflip

Fileemd_41097_half_map_1.map
AnnotationHalf map A, zflip
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B, zflip

Fileemd_41097_half_map_2.map
AnnotationHalf map B, zflip
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Smc5/6 5mer

EntireName: Smc5/6 5mer
Components
  • Complex: Smc5/6 5mer
    • Protein or peptide: Structural maintenance of chromosomes protein 6
    • Protein or peptide: Non-structural maintenance of chromosome element 5
    • Protein or peptide: DNA repair protein KRE29

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Supramolecule #1: Smc5/6 5mer

SupramoleculeName: Smc5/6 5mer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
Molecular weightTheoretical: 234 kDa/nm

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Macromolecule #1: Structural maintenance of chromosomes protein 6

MacromoleculeName: Structural maintenance of chromosomes protein 6 / type: protein_or_peptide / ID: 1
Details: the visible residues from Smc6 (with E1048Q mutation)in the structure are the N terminal part of 76-271aa and C terminal part of 949-1100aa.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
Molecular weightTheoretical: 128.198742 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: MISTTISGKR PIEQVDDELL SLTAQQENEE QQQQRKRRRH QFAPMTQFNS NTLDEDSGFR SSSDVATADQ DNFLEESPSG YIKKVILRN FMCHEHFELE LGSRLNFIVG NNGSGKSAIL TAITIGLGAK ASETNRGSSL KDLIREGCYS AKIILHLDNS K YGAYQQGI ...String:
MISTTISGKR PIEQVDDELL SLTAQQENEE QQQQRKRRRH QFAPMTQFNS NTLDEDSGFR SSSDVATADQ DNFLEESPSG YIKKVILRN FMCHEHFELE LGSRLNFIVG NNGSGKSAIL TAITIGLGAK ASETNRGSSL KDLIREGCYS AKIILHLDNS K YGAYQQGI FGNEIIVERI IKRDGPASFS LRSENGKEIS NKKKDIQTVV DYFSVPVSNP MCFLSQDAAR SFLTASTSQD KY SHFMKGT LLQEITENLL YASAIHDSAQ ENMALHLENL KSLKAEYEDA KKLLRELNQT SDLNERKMLL QAKSLWIDVA HNT DACKNL ENEISGIQQK VDEVTEKIRN RQEKIERYTS DGTTIEAQID AKVIYVNEKD SEHQNARELL RDVKSRFEKE KSNQ AEAQS NIDQGRKKVD ALNKTIAHLE EELTKEMGGD KDQMRQELEQ LEKANEKLRE VNNSLVVSLQ DVKNEERDIQ HERES ELRT ISRSIQNKKV ELQNIAKGND TFLMNFDRNM DRLLRTIEQR KNEFETPAIG PLGSLVTIRK GFEKWTRSIQ RAISSS LNA FVVSNPKDNR LFRDIMRSCG IRSNIPIVTY CLSQFDYSKG RAHGNYPTIV DALEFSKPEI ECLFVDLSRI ERIVLIE DK NEARNFLQRN PVNVNMALSL RDRRSGFQLS GGYRLDTVTY QDKIRLKVNS SSDNGTQYLK DLIEQETKEL QNIRDRYE E KLSEVRSRLK EIDGRLKSTK NEMRKTNFRM TELKMNVGKV VDTGILNSKI NERKNQEQAI ASYEAAKEEL GLKIEQIAQ EAQPIKEQYD STKLALVEAQ DELQQLKEDI NSRQSKIQKY KDDTIYYEDK KKVYLENIKK IEVNVAALKE GIQRQIQNAC AFCSKERIE NVDLPDTQEE IKRELDKVSR MIQKAEKSLG LSQEEVIALF EKCRNKYKEG QKKYMEIDEA LNRLHNSLKA R DQNYKNAE KGTCFDADMD FRASLKVRKF SGNLSFIKDT KSLEIYILTT NDEKARNVDT LSGGEKSFSQ MALLLATWKP MR SRIIALD QFDVFMDQVN RKIGTTLIVK KLKDIARTQT IIITPQDIGK IADIDSSGVS IHRMRDPERQ NNSNFYN

UniProtKB: Structural maintenance of chromosomes protein 6

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Macromolecule #2: Non-structural maintenance of chromosome element 5

MacromoleculeName: Non-structural maintenance of chromosome element 5 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
Molecular weightTheoretical: 64.079609 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: MDGALINSVL YVSPRNGAHY FVELTEKHLL AFEMLNSMCL LENYDHVLLF LECQFGKSHN LAVIPFDIIL VLFTLSTLSE YYKEPILRA NDPYNTSRET LSRRALKLLQ KYLAILKEFD SEQYNLYDLE LLRCQFFLAI DTLTPKKQKW GFDRFRRTKS E SGVTYRQN ...String:
MDGALINSVL YVSPRNGAHY FVELTEKHLL AFEMLNSMCL LENYDHVLLF LECQFGKSHN LAVIPFDIIL VLFTLSTLSE YYKEPILRA NDPYNTSRET LSRRALKLLQ KYLAILKEFD SEQYNLYDLE LLRCQFFLAI DTLTPKKQKW GFDRFRRTKS E SGVTYRQN ASVDPELDQA KTFKNPYRSY ISCLEQRNTI LGNRLLNLKL NEPGEFINMI LWTLSNSLQE STPLFLSSHE IW MPLLEIL IDLFSCRQDY FIQHEVAQNV SKSLFVQRLS ESPLAVFFES LNTRNFANRF SEYVFLNCDY KLPSDNYATP VHP VYNGEN TIVDTYIPTI KCSPLYKSQK SLALRRKLIG SCFKLLLRVP DGHRLITPRI VADDVIQGIS RTLASFNDIL QFKK FFMTE NLSQESYFIP LLAEGTLSEI LKDTQECVVI LTLVENLSDG VSFCNEVIGL VKSKCFAFTE QCSQASYEEA VLNIE KCDV CLLVLLRYLL HLIGTEAILD AKEQLEMLHA IEKNDSGRRQ WAKALNLGND PPLLYPIVSQ MFGVHDKSVI IE

UniProtKB: Non-structural maintenance of chromosome element 5

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Macromolecule #3: DNA repair protein KRE29

MacromoleculeName: DNA repair protein KRE29 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
Molecular weightTheoretical: 53.836758 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: MGSVNSSPNE EFETVPDSQI SGFDSPLIPT SVGSYFRDDD DDEKVHPNFI SDPENDSLNS DEEFSSLENS DLNLSGAKAE SGDDFDPIL KRTIISKRKA PSNNEDEEIV KTPRKLVNYV PLKIFNLGDS FDDTITTTVA KLQDLKKEIL DSPRSNKSIV I TSNTVAKS ...String:
MGSVNSSPNE EFETVPDSQI SGFDSPLIPT SVGSYFRDDD DDEKVHPNFI SDPENDSLNS DEEFSSLENS DLNLSGAKAE SGDDFDPIL KRTIISKRKA PSNNEDEEIV KTPRKLVNYV PLKIFNLGDS FDDTITTTVA KLQDLKKEIL DSPRSNKSIV I TSNTVAKS ELQKSIKFSG SIPEIYLDVV TKETISDKYK DWHFISKNCH YEQLMDLEMK DTAYSFLFGS SRSQGKVPEF VH LKCPSIT NLLVLFGVNQ EKCNSLKINY EKKENSRYDN LCTIFPVNKM LKFLMYFYSD DDNDDVREFF LKAFICLILD RKV FNAMES DHRLCFKVLE LFNEAHFINS YFEIVDKNDF FLHYRLLQIF PHLQSALLRR RFSEKQGRTE TIQQNIIKEF NEFF DCKNY KNLLYFILTM YGSKFIPFGP KCQVTEYFKD CILDISNETT NDVEISILKG ILNLFSKIR

UniProtKB: DNA repair protein KRE29

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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

Concentration0.31 mg/mL
BufferpH: 7.5 / Details: 25 mM Hepes, pH 7.5, 250 mM NaCl, 1 mM DTT
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 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 opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
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
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 219202

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