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- EMDB-54602: Cryo-EM structure of the Saccharomyces cerevisiae KMN junction co... -

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Basic information

Entry
Database: EMDB / ID: EMD-54602
TitleCryo-EM structure of the Saccharomyces cerevisiae KMN junction complex containing the Mis12c(Mtw1c) head 2 domain
Map data
Sample
  • Complex: Saccharomyces cerevisiae outer kinetochore KMN junction complex
    • Complex: Saccharomyces cerevisiae outer kinetochore Mis12c(Mtw1c) subcomplex
      • Protein or peptide: Kinetochore-associated protein NNF1
      • Protein or peptide: Kinetochore-associated protein MTW1
      • Protein or peptide: Kinetochore-associated protein DSN1
      • Protein or peptide: Kinetochore-associated protein NSL1
    • Complex: Saccharomyces cerevisiae outer kinetochore Knl1c subcomplex
      • Protein or peptide: Outer kinetochore KNL1 complex subunit KRE28
      • Protein or peptide: Outer kinetochore KNL1 complex subunit SPC105
    • Complex: Saccharomyces cerevisiae outer kinetochore Ndc80c
      • Protein or peptide: Kinetochore protein SPC24
      • Protein or peptide: Kinetochore protein SPC25
KeywordsKinetochore / chromosome segregation / mitosis / cell cycle
Function / homology
Function and homology information


regulation of meiosis I spindle assembly checkpoint / Knl1/Spc105 complex / positive regulation of meiosis I spindle assembly checkpoint / homologous chromosome orientation in meiotic metaphase I / centromere clustering / MIS12/MIND type complex / Ndc80 complex / mitotic sister chromatid biorientation / spindle attachment to meiosis I kinetochore / sister chromatid biorientation ...regulation of meiosis I spindle assembly checkpoint / Knl1/Spc105 complex / positive regulation of meiosis I spindle assembly checkpoint / homologous chromosome orientation in meiotic metaphase I / centromere clustering / MIS12/MIND type complex / Ndc80 complex / mitotic sister chromatid biorientation / spindle attachment to meiosis I kinetochore / sister chromatid biorientation / attachment of spindle microtubules to kinetochore / meiotic sister chromatid cohesion, centromeric / kinetochore assembly / outer kinetochore / protein localization to kinetochore / mitotic spindle assembly checkpoint signaling / mitotic sister chromatid segregation / chromosome, centromeric region / Neutrophil degranulation / chromosome segregation / kinetochore / spindle pole / microtubule binding / cell division / mitochondrion / identical protein binding / nucleus
Similarity search - Function
Spindle pole body component Kre28 / Spindle pole body component Kre28 / Spc7 kinetochore protein domain / Spc105/Spc7 / Spc7 kinetochore protein / Spc7 kinetochore protein / Kinetochore-associated protein Nnf1 / Spc24, Fungi, globular domain superfamily / Knl1, C-terminal RWD domain / Knl1 RWD C-terminal domain ...Spindle pole body component Kre28 / Spindle pole body component Kre28 / Spc7 kinetochore protein domain / Spc105/Spc7 / Spc7 kinetochore protein / Spc7 kinetochore protein / Kinetochore-associated protein Nnf1 / Spc24, Fungi, globular domain superfamily / Knl1, C-terminal RWD domain / Knl1 RWD C-terminal domain / Kinetochore Mis14/Nsl1 / Kinetochore protein Mis14 like / Nuclear MIS12/MIND complex subunit PMF1/Nnf1 / Centromere protein Mis12 / Nnf1 / Mis12 protein / Kinetochore-associated protein Dsn1/Mis13 / Mis12-Mtw1 protein family / Kinetochore-Ndc80 subunit Spc24 / Spc24 subunit of Ndc80 / EF-Hand 1, calcium-binding site
Similarity search - Domain/homology
Kinetochore-associated protein MTW1 / Kinetochore protein SPC25 / Kinetochore-associated protein DSN1 / Kinetochore-associated protein NNF1 / Outer kinetochore KNL1 complex subunit SPC105 / Outer kinetochore KNL1 complex subunit KRE28 / Kinetochore protein SPC24 / Kinetochore-associated protein NSL1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Saccharomyces cerevisiae S288C (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.2 Å
AuthorsTurner NN / Barford D
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Cancer Research UKC576/A25675 United Kingdom
Cancer Research UKC576/A14109 United Kingdom
UK Research and Innovation (UKRI)MC_UP_1201/6 United Kingdom
CitationJournal: J Cell Biol / Year: 2026
Title: Assembly and phosphoregulatory mechanisms of the budding yeast outer kinetochore KMN complex.
Authors: Noah N Turner / Ziguo Zhang / Jing Yang / Kyle W Muir / Stephen H McLaughlin / Tomos Morgan / David Barford /
Abstract: During mitosis and meiosis, kinetochores mediate interactions between chromosomes and spindle microtubules. Kinetochores are multi-megadalton protein complexes essential for chromosome segregation; ...During mitosis and meiosis, kinetochores mediate interactions between chromosomes and spindle microtubules. Kinetochores are multi-megadalton protein complexes essential for chromosome segregation; however, recent structural, functional, and evolutionary studies have revealed divergent mechanisms of kinetochore assembly. Here, we use cryo-EM to understand the structural mechanisms by which the budding yeast microtubule-binding outer kinetochore KMN complex assembles, and how its interactions with the centromere-binding inner kinetochore are regulated. The KMN complex comprises three subcomplexes: Knl1c, Mis12cMtw1c, and Ndc80c. We show how C-terminal motifs of the Mis12cMtw1c subunits Dsn1, Mis12Mtw1, and Nnf1 bind Knl1c and Ndc80c. At the opposite end of the Mis12cMtw1c stalk, an N-terminal auto-inhibitory segment of Dsn1 (Dsn1AI) folds into two α-helices that engage the Mis12cMtw1c head 1 domain, thereby occluding binding sites for the inner kinetochore subunits CENP-CMif2 and CENP-UAme1, reducing their affinity for Mis12cMtw1. Our structure reveals how Aurora BIpl1 phosphorylation of Dsn1AI would release this auto-inhibition to substantially strengthen preexisting connections between the inner and outer kinetochore.
History
DepositionJul 29, 2025-
Header (metadata) releaseApr 8, 2026-
Map releaseApr 8, 2026-
UpdateApr 22, 2026-
Current statusApr 22, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_54602.png

Downloads & links

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Map

FileDownload / File: emd_54602.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.65 Å/pix.
x 192 pix.
= 316.8 Å		1.65 Å/pix.
x 192 pix.
= 316.8 Å		1.65 Å/pix.
x 192 pix.
= 316.8 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.028
Minimum - Maximum-0.039500374 - 0.09978225
Average (Standard dev.)0.0001061669 (±0.0036640915)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 316.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_54602_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_54602_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : Saccharomyces cerevisiae outer kinetochore KMN junction complex

EntireName: Saccharomyces cerevisiae outer kinetochore KMN junction complex
Components
  • Complex: Saccharomyces cerevisiae outer kinetochore KMN junction complex
    • Complex: Saccharomyces cerevisiae outer kinetochore Mis12c(Mtw1c) subcomplex
      • Protein or peptide: Kinetochore-associated protein NNF1
      • Protein or peptide: Kinetochore-associated protein MTW1
      • Protein or peptide: Kinetochore-associated protein DSN1
      • Protein or peptide: Kinetochore-associated protein NSL1
    • Complex: Saccharomyces cerevisiae outer kinetochore Knl1c subcomplex
      • Protein or peptide: Outer kinetochore KNL1 complex subunit KRE28
      • Protein or peptide: Outer kinetochore KNL1 complex subunit SPC105
    • Complex: Saccharomyces cerevisiae outer kinetochore Ndc80c
      • Protein or peptide: Kinetochore protein SPC24
      • Protein or peptide: Kinetochore protein SPC25

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Supramolecule #1: Saccharomyces cerevisiae outer kinetochore KMN junction complex

SupramoleculeName: Saccharomyces cerevisiae outer kinetochore KMN junction complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Imaged sample also contained Saccharomyces cerevisiae kinetochore protein NDC80 (UniProtKB accession P40460), and Saccharomyces cerevisiae kinetochore protein NUF2 (UniProtKB accession ...Details: Imaged sample also contained Saccharomyces cerevisiae kinetochore protein NDC80 (UniProtKB accession P40460), and Saccharomyces cerevisiae kinetochore protein NUF2 (UniProtKB accession P33895) with a C-terminal HRV-3C protease cleavage site and twin-strep-tag II affinity tag.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: 288c
Molecular weightTheoretical: 187.080 kDa/nm

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Supramolecule #2: Saccharomyces cerevisiae outer kinetochore Mis12c(Mtw1c) subcomplex

SupramoleculeName: Saccharomyces cerevisiae outer kinetochore Mis12c(Mtw1c) subcomplex
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2, #5, #8
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: 288c

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Supramolecule #3: Saccharomyces cerevisiae outer kinetochore Knl1c subcomplex

SupramoleculeName: Saccharomyces cerevisiae outer kinetochore Knl1c subcomplex
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Details: N-terminal truncation of Knl1(Spc105) residues 1-444
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: 288c

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Supramolecule #4: Saccharomyces cerevisiae outer kinetochore Ndc80c

SupramoleculeName: Saccharomyces cerevisiae outer kinetochore Ndc80c / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #6-#7
Details: Also contains Saccharomyces cerevisiae kinetochore protein NDC80 (UniProtKB accession P40460), and Saccharomyces cerevisiae kinetochore protein NUF2 (UniProtKB accession P33895) with a C- ...Details: Also contains Saccharomyces cerevisiae kinetochore protein NDC80 (UniProtKB accession P40460), and Saccharomyces cerevisiae kinetochore protein NUF2 (UniProtKB accession P33895) with a C-terminal HRV-3C protease cleavage site and twin-strep-tag II affinity tag that were present in the imaged sample.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: 288c

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Macromolecule #1: Kinetochore-associated protein NNF1

MacromoleculeName: Kinetochore-associated protein NNF1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 23.668541 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MVNSHGIRYI RLKQVFNRAL DQSISKLQSW DKVSSCFPQY VNSKQGAINV ANCQRQLTEF WTELCQREFK EIMEERNVEQ KLNELDELI LEAKERYTDR DQDEVNKGPA IDELSSKELV ECHLYSQRMH AIHEIDERLA KVNEMNDQLA QELKDLETQV E VEKNEIGK ...String:
MVNSHGIRYI RLKQVFNRAL DQSISKLQSW DKVSSCFPQY VNSKQGAINV ANCQRQLTEF WTELCQREFK EIMEERNVEQ KLNELDELI LEAKERYTDR DQDEVNKGPA IDELSSKELV ECHLYSQRMH AIHEIDERLA KVNEMNDQLA QELKDLETQV E VEKNEIGK MYDEYLGSHT DQPANVLLVQ SLNDMVLELK ENY

UniProtKB: Kinetochore-associated protein NNF1

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Macromolecule #2: Kinetochore-associated protein MTW1

MacromoleculeName: Kinetochore-associated protein MTW1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 33.29118 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSAPTMRSTS ILTEHLGYPP ISLVDDIINA VNEIMYKCTA AMEKYLLSKS KIGEEDYGEE IKSGVAKLES LLENSVDKNF DKLELYVLR NVLRIPEEYL DANVFRLENQ KDLVIVDENE LKKSEEKLRE KVNDVELAFK KNEMLLKRVT KVKRLLFTIR G FKQKLNEL ...String:
MSAPTMRSTS ILTEHLGYPP ISLVDDIINA VNEIMYKCTA AMEKYLLSKS KIGEEDYGEE IKSGVAKLES LLENSVDKNF DKLELYVLR NVLRIPEEYL DANVFRLENQ KDLVIVDENE LKKSEEKLRE KVNDVELAFK KNEMLLKRVT KVKRLLFTIR G FKQKLNEL LKCKDDVQLQ KILESLKPID DTMTLLTDSL RKLYVDSEST SSTEEVEALL QRLKTNGKQN NKDFRTRYID IR TNNVLRK LGLLGDKEDE KQSAKPDART QAGDIVSIDI EEPQLDLLDD VL

UniProtKB: Kinetochore-associated protein MTW1

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Macromolecule #3: Outer kinetochore KNL1 complex subunit KRE28

MacromoleculeName: Outer kinetochore KNL1 complex subunit KRE28 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 48.468273 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDTGSASIKD YETVLTDIED SIAVSSEEVL NNQELRLKNT LHEITSSILA INEENKFVNP LRNDESLDVE GKEVFVNPKI LSAKIKEFN KLMELLKLTY LEQETLDYFF RFTLSSTKPL QLDSEKDPQF VKLNERVNDL KEEISNVQES KIEQIKAEIQ E TGHNFAER ...String:
MDTGSASIKD YETVLTDIED SIAVSSEEVL NNQELRLKNT LHEITSSILA INEENKFVNP LRNDESLDVE GKEVFVNPKI LSAKIKEFN KLMELLKLTY LEQETLDYFF RFTLSSTKPL QLDSEKDPQF VKLNERVNDL KEEISNVQES KIEQIKAEIQ E TGHNFAER QDLINELYLE ATGDIENCWD SLNELKNLTN KEDKNMMGEK DTILNSSDSD DFVEETYTNW QKLLFLQKQN QR LTKELKE MHEVKNQIIR KGEQSKKEDS GHLMANESEL CQSINLLTKF WEKHFLLKGS KTTILNFEIF TQLGKVQFEI KDM QYIIAI SLSDLKRPMI KDITILQKAG GNIVTDIEAS SKFNNKYRNN TKVQIFEVMD DIISELTNEE NLYFQSWSHP QFEK GGGSG GGSGGGSWSH PQFEK

UniProtKB: Outer kinetochore KNL1 complex subunit KRE28

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Macromolecule #4: Outer kinetochore KNL1 complex subunit SPC105

MacromoleculeName: Outer kinetochore KNL1 complex subunit SPC105 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 55.581047 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSGIALPTQD YTLREFINEV GVGFLDTKLI DDLDKKVNFP LNSFNFVENQ RIDNVFSAFY IDIPILEVEA FRCKELWRSI NESKDKFKD FEAQIDKSHP PLLLQEYFSS DEKMKQLMRD QLQLVKGYSK LEAAMEWYEW RKKQLNGLEL ILAENLNTLK R EYEKLNEE ...String:
MSGIALPTQD YTLREFINEV GVGFLDTKLI DDLDKKVNFP LNSFNFVENQ RIDNVFSAFY IDIPILEVEA FRCKELWRSI NESKDKFKD FEAQIDKSHP PLLLQEYFSS DEKMKQLMRD QLQLVKGYSK LEAAMEWYEW RKKQLNGLEL ILAENLNTLK R EYEKLNEE VEKVNSIRGK IRKLNEAIKE EIRSLKNLPS DSYKPTLMNR IKIEAFKQEL MEHSISLSSS NDFTQEMRSL KL AIAKKSN DILTLRSEVA SIDKKIEKRK LFTRFDLPKL RDTLKILESL TGVRFLKFSK ATLSIAFLQL DDLRVDINLA NFK NNPLSS MKVMNDSNND DMSYHLFTML LKNVEAEHQD SMLSNLFFAM KKWRPLLKYI KLLKLLFPVK ITQTEEEEAL LQFK DYDRR NKTAFFYVIS LVSFAQGVFS ENGQIPMKVH ISTQQDYSPS REVLSDRITH KISGVLPSFT KSRIHLEFT

UniProtKB: Outer kinetochore KNL1 complex subunit SPC105

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Macromolecule #5: Kinetochore-associated protein DSN1

MacromoleculeName: Kinetochore-associated protein DSN1 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 65.777023 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSLEPTQTVS GTPPMLHQRT HKQVYPLRME TIPILESDSK ATLQSNEPTQ KDEEETEYFE NKQSVSNLSP DLKFKRHKNK HIQGFPTLG ERLDNLQDIK KAKRVENFNS SAPIADDNHS GDATANATAN ATANATANVN ASAMPAPYMP YYYYYHPMNA P TPAMIPYP ...String:
MSLEPTQTVS GTPPMLHQRT HKQVYPLRME TIPILESDSK ATLQSNEPTQ KDEEETEYFE NKQSVSNLSP DLKFKRHKNK HIQGFPTLG ERLDNLQDIK KAKRVENFNS SAPIADDNHS GDATANATAN ATANATANVN ASAMPAPYMP YYYYYHPMNA P TPAMIPYP GSPMHSIMPN SSLQPFYSQP TAAGGPDMTT PQNISSSQQL LPAPQLFPYG SFHQQQLQQP HYIQRTRERK KS IGSQRGR RLSMLASQAN GGSTIISPHK DIPEEDFYTV VGNASFGKNL QIRQLFNWCL MRSLHKLELK AKNQEEEGEL EHL TKKSKL ESTKAETDYV DPKRLAMVII KEFVDDLKKD HIAIDWEDEE KYEDEDEEKI LDNTENYDDT ELRQLFQEND DDDD DDDEV DYSEIQRSRR KFSERRKALP KEPKKLLPNS KNVENTKNLS ILTSKVNAIK NEVKEWAVTL DTSRPDLEWQ ELTSF SSQP LEPLSDTEEP DLAIADVETK LETKVDELRY QSHILNSHSL ALNEITNSKV NKLNIETMRK ISSETDDDHS QVINPQ QLL KGLSLSFSKK LDL

UniProtKB: Kinetochore-associated protein DSN1

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Macromolecule #6: Kinetochore protein SPC24

MacromoleculeName: Kinetochore protein SPC24 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 24.639814 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSQKDNLLDN PVEFLKEVRE SFDIQQDVDA MKRIRHDLDV IKEESEARIS KEHSKVSESN KKLNAERINV AKLEGDLEYT NEESNEFGS KDELVKLLKD LDGLERNIVS LRSELDEKMK LYLKDSEIIS TPNGSKIKAK VIEPELEEQS AVTPEANENI L KLKLYRSL ...String:
MSQKDNLLDN PVEFLKEVRE SFDIQQDVDA MKRIRHDLDV IKEESEARIS KEHSKVSESN KKLNAERINV AKLEGDLEYT NEESNEFGS KDELVKLLKD LDGLERNIVS LRSELDEKMK LYLKDSEIIS TPNGSKIKAK VIEPELEEQS AVTPEANENI L KLKLYRSL GVILDLENDQ VLINRKNDGN IDILPLDNNL SDFYKTKYIW ERLGK

UniProtKB: Kinetochore protein SPC24

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Macromolecule #7: Kinetochore protein SPC25

MacromoleculeName: Kinetochore protein SPC25 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 25.280326 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MASIDAFSDL ERRMDGFQKD VAQVLARQQN HARQQLQQFQ AEMRQLHNQH QHLIDELQRL ATQRTALQQQ IHAAQQATNT TREQWRSYH ERESELSRRQ STLAAQSREL DSLLQQRGKE CVQLRARWAA QSGNDAAEVA LYERLLQLRV LPGASDVHDV R FVFGDDSR ...String:
MASIDAFSDL ERRMDGFQKD VAQVLARQQN HARQQLQQFQ AEMRQLHNQH QHLIDELQRL ATQRTALQQQ IHAAQQATNT TREQWRSYH ERESELSRRQ STLAAQSREL DSLLQQRGKE CVQLRARWAA QSGNDAAEVA LYERLLQLRV LPGASDVHDV R FVFGDDSR CWIEVAMHGD HVIGNSHPAL DPKSRATLEH VLTVQGDLAA FLVVARDMLL ASL

UniProtKB: Kinetochore protein SPC25

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Macromolecule #8: Kinetochore-associated protein NSL1

MacromoleculeName: Kinetochore-associated protein NSL1 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 25.448398 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSQGQSKKLD VTVEQLRSIY HQFHDILEEK TDLHLPKKEY DDDAVRREVQ IQLQEFLLSA MTMASKSLEV VNADTVGKTV KQLIMESQE KYMEPFDLDL NEQVRKMYQE WEDETVKVAQ LRQTGPAKIN EVYNNSKDEY LAQLDGRIGV LQARMMQQQS A DHDDSTDD ...String:
MSQGQSKKLD VTVEQLRSIY HQFHDILEEK TDLHLPKKEY DDDAVRREVQ IQLQEFLLSA MTMASKSLEV VNADTVGKTV KQLIMESQE KYMEPFDLDL NEQVRKMYQE WEDETVKVAQ LRQTGPAKIN EVYNNSKDEY LAQLDGRIGV LQARMMQQQS A DHDDSTDD ADDHINWEHI KQDYVASLNE LYQTQQDLPK VRYNVEKVKR LMDFLEED

UniProtKB: Kinetochore-associated protein NSL1

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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

Concentration0.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPES
150.0 mMSodium chlorideNaCl
1.0 mMTCEP
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Pressure: 0.02 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
DetailsCrosslinked using glutaraldehyde with GraFix methodology. Crosslinker was quenched with 20 mM Tris pH 7.5.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 26390 / Average exposure time: 1.66 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2592458
Details: Selected using Topaz particle picker trained on manually picked micrographs
CTF correctionSoftware - Name: CTFFIND (ver. 4.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 7.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5) / Details: Refined using Blush regularization / Number images used: 18160
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5)
Final 3D classificationNumber classes: 4 / Avg.num./class: 80269
Details: Focussed classification using Blush regularization.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

9s4q

chain_id: Mt, residue_range: 1-289, source_name: PDB, initial_model_type: experimental model

9s4q

chain_id: Nn, residue_range: 1-201, source_name: PDB, initial_model_type: experimental model

9s4q

chain_id: Ds, residue_range: 435-576, source_name: PDB, initial_model_type: experimental model

9s53

chain_id: Ds, residue_range: 229-365, source_name: PDB, initial_model_type: experimental model

9s4q

chain_id: Ns, residue_range: 97-216, source_name: PDB, initial_model_type: experimental model

9s53

chain_id: Ns, residue_range: 2-96, source_name: PDB, initial_model_type: experimental model

9s4q

chain_id: Kl, residue_range: 719-917, source_name: PDB, initial_model_type: experimental model

9s4q

chain_id: Zw, residue_range: 280-385, source_name: PDB, initial_model_type: experimental model

9s4q

chain_id: 24, residue_range: 143-213, source_name: PDB, initial_model_type: experimental model

9s4q

chain_id: 25, residue_range: 124-221, source_name: PDB, initial_model_type: experimental model
SoftwareName: UCSF ChimeraX
RefinementProtocol: RIGID BODY FIT
Output model

PDB-9s5n:
Cryo-EM structure of the Saccharomyces cerevisiae KMN junction complex containing the Mis12c(Mtw1c) head 2 domain

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