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- EMDB-30237: The cryo-EM structure of CENP-A nucleosome in complex with CENP-C... -

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Basic information

Entry
Database: EMDB / ID: EMD-30237
TitleThe cryo-EM structure of CENP-A nucleosome in complex with CENP-C peptide and CENP-N N-terminal domain
Map dataggCENP-A nucleosome in complex with CENP-C peptide (643-683) and CENP-N (1-212)
Sample
  • Complex: CENP-A nucleosome in complex with CENP-C peptide and CNEP-N N-terminal domain
    • Complex: Histone H3,Histone H3-like centromeric protein A
      • Protein or peptide: Histone H3,Histone H3-like centromeric protein A
    • Complex: Histone H4
      • Protein or peptide: Histone H4
    • Complex: Histone H2A type 1-B/E
      • Protein or peptide: Histone H2A type 1-B/E
    • Complex: Histone H2B type 2-E
      • Protein or peptide: Histone H2B type 2-E
    • Complex: DNA
      • DNA: DNA (145-mer)
      • DNA: DNA (145-mer)
    • Complex: CENP-C
      • Protein or peptide: CENP-C
    • Complex: Maltodextrin-binding protein,Centromere protein N
      • Protein or peptide: Maltodextrin-binding protein,Centromere protein N
KeywordsCENP-A nucleosome / CENP-C / CENP-N / complex / kinetochore / NUCLEAR PROTEIN / CELL CYCLE-DNA complex
Function / homology
Function and homology information


kinetochore => GO:0000776 / Interleukin-7 signaling / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Chromatin modifying enzymes / Resolution of Sister Chromatid Cohesion / EML4 and NUDC in mitotic spindle formation / RHO GTPases Activate Formins / Formation of the beta-catenin:TCF transactivating complex / PRC2 methylates histones and DNA / Oxidative Stress Induced Senescence ...kinetochore => GO:0000776 / Interleukin-7 signaling / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Chromatin modifying enzymes / Resolution of Sister Chromatid Cohesion / EML4 and NUDC in mitotic spindle formation / RHO GTPases Activate Formins / Formation of the beta-catenin:TCF transactivating complex / PRC2 methylates histones and DNA / Oxidative Stress Induced Senescence / HDACs deacetylate histones / HATs acetylate histones / Transcriptional regulation by small RNAs / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / Assembly of the ORC complex at the origin of replication / RNA Polymerase I Promoter Opening / RNA Polymerase I Promoter Escape / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Estrogen-dependent gene expression / Regulation of endogenous retroelements by KRAB-ZFP proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / B-WICH complex positively regulates rRNA expression / Separation of Sister Chromatids / Deposition of new CENPA-containing nucleosomes at the centromere / Factors involved in megakaryocyte development and platelet production / spindle attachment to meiosis I kinetochore / centromeric DNA binding / CENP-A containing chromatin assembly / protein localization to chromosome, centromeric region / kinetochore assembly / attachment of mitotic spindle microtubules to kinetochore / condensed chromosome, centromeric region / detection of maltose stimulus / maltose transport complex / establishment of mitotic spindle orientation / maltose binding / carbohydrate transport / maltose transport / maltodextrin transmembrane transport / mitotic cytokinesis / chromosome, centromeric region / carbohydrate transmembrane transporter activity / negative regulation of megakaryocyte differentiation / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / protein localization to CENP-A containing chromatin / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / Packaging Of Telomere Ends / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Deposition of new CENPA-containing nucleosomes at the centromere / nucleosomal DNA binding / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Inhibition of DNA recombination at telomere / telomere organization / Meiotic synapsis / RNA Polymerase I Promoter Opening / Assembly of the ORC complex at the origin of replication / SUMOylation of chromatin organization proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / ATP-binding cassette (ABC) transporter complex / DNA methylation / cell chemotaxis / Condensation of Prophase Chromosomes / SIRT1 negatively regulates rRNA expression / Chromatin modifications during the maternal to zygotic transition (MZT) / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / HCMV Late Events / innate immune response in mucosa / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / chromosome segregation / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / HDACs deacetylate histones / Nonhomologous End-Joining (NHEJ) / RNA Polymerase I Promoter Escape / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / NoRC negatively regulates rRNA expression / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / G2/M DNA damage checkpoint / HDMs demethylate histones / B-WICH complex positively regulates rRNA expression / DNA Damage/Telomere Stress Induced Senescence / heterochromatin formation / PKMTs methylate histone lysines / Metalloprotease DUBs / Meiotic recombination / kinetochore / Pre-NOTCH Transcription and Translation / RMTs methylate histone arginines / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / Transcriptional regulation of granulopoiesis / structural constituent of chromatin / UCH proteinases / antimicrobial humoral immune response mediated by antimicrobial peptide
Similarity search - Function
: / Kinetochore assembly subunit CENP-C, N-terminal domain / Kinetochore assembly subunit CENP-C N-terminal / Mif2/CENP-C cupin domain / Centromere protein C/Mif2/cnp3 / Mif2/CENP-C like / Centromere protein Chl4/mis15/CENP-N / Kinetochore protein CHL4 like / RmlC-like cupin domain superfamily / Maltose/Cyclodextrin ABC transporter, substrate-binding protein ...: / Kinetochore assembly subunit CENP-C, N-terminal domain / Kinetochore assembly subunit CENP-C N-terminal / Mif2/CENP-C cupin domain / Centromere protein C/Mif2/cnp3 / Mif2/CENP-C like / Centromere protein Chl4/mis15/CENP-N / Kinetochore protein CHL4 like / RmlC-like cupin domain superfamily / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / RmlC-like jelly roll fold / Histone H2A / Histone 2A / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold
Similarity search - Domain/homology
Uncharacterized protein / CENP-C / Histone H2A type 1-B/E / Maltose/maltodextrin-binding periplasmic protein / Histone H4 / Histone H3.2 / Histone H2B type 2-E / Centromere protein N / Histone H3-like centromeric protein A
Similarity search - Component
Biological speciesEctocarpus siliculosus (eukaryote) / Homo sapiens (human) / Escherichia coli (strain B / BL21-DE3) (bacteria) / Gallus gallus (chicken) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsAriyoshi M / Makino F
Funding support Japan, 3 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)25221106 Japan
Japan Agency for Medical Research and Development (AMED)0101117 Japan
Japan Agency for Medical Research and Development (AMED)0101020 Japan
CitationJournal: EMBO J / Year: 2021
Title: Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C.
Authors: Mariko Ariyoshi / Fumiaki Makino / Reito Watanabe / Reiko Nakagawa / Takayuki Kato / Keiichi Namba / Yasuhiro Arimura / Risa Fujita / Hitoshi Kurumizaka / Ei-Ichi Okumura / Masatoshi Hara / Tatsuo Fukagawa /
Abstract: The CENP-A nucleosome is a key structure for kinetochore assembly. Once the CENP-A nucleosome is established in the centromere, additional proteins recognize the CENP-A nucleosome to form a ...The CENP-A nucleosome is a key structure for kinetochore assembly. Once the CENP-A nucleosome is established in the centromere, additional proteins recognize the CENP-A nucleosome to form a kinetochore. CENP-C and CENP-N are CENP-A binding proteins. We previously demonstrated that vertebrate CENP-C binding to the CENP-A nucleosome is regulated by CDK1-mediated CENP-C phosphorylation. However, it is still unknown how the phosphorylation of CENP-C regulates its binding to CENP-A. It is also not completely understood how and whether CENP-C and CENP-N act together on the CENP-A nucleosome. Here, using cryo-electron microscopy (cryo-EM) in combination with biochemical approaches, we reveal a stable CENP-A nucleosome-binding mode of CENP-C through unique regions. The chicken CENP-C structure bound to the CENP-A nucleosome is stabilized by an intramolecular link through the phosphorylated CENP-C residue. The stable CENP-A-CENP-C complex excludes CENP-N from the CENP-A nucleosome. These findings provide mechanistic insights into the dynamic kinetochore assembly regulated by CDK1-mediated CENP-C phosphorylation.
History
DepositionApr 20, 2020-
Header (metadata) releaseFeb 10, 2021-
Map releaseFeb 10, 2021-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bxt
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30237.png

Downloads & links

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Map

FileDownload / File: emd_30237.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationggCENP-A nucleosome in complex with CENP-C peptide (643-683) and CENP-N (1-212)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.47 Å/pix.
x 192 pix.
= 282.24 Å		1.47 Å/pix.
x 192 pix.
= 282.24 Å		1.47 Å/pix.
x 192 pix.
= 282.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.47 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.035
Minimum - Maximum-0.077114105 - 0.13460799
Average (Standard dev.)0.00033561292 (±0.005340674)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 282.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.471.471.47
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z282.240282.240282.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0770.1350.000

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Supplemental data

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Mask #1

Fileemd_30237_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 1st half map file for ggCENP-A nucleosome in...

Fileemd_30237_half_map_1.map
Annotation1st half map file for ggCENP-A nucleosome in complex with CENP-C peptide (643-683) and CENP-N (1-212)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 1st half map file for ggCENP-A nucleosome in...

Fileemd_30237_half_map_2.map
Annotation1st half map file for ggCENP-A nucleosome in complex with CENP-C peptide (643-683) and CENP-N (1-212)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CENP-A nucleosome in complex with CENP-C peptide and CNEP-N N-ter...

EntireName: CENP-A nucleosome in complex with CENP-C peptide and CNEP-N N-terminal domain
Components
  • Complex: CENP-A nucleosome in complex with CENP-C peptide and CNEP-N N-terminal domain
    • Complex: Histone H3,Histone H3-like centromeric protein A
      • Protein or peptide: Histone H3,Histone H3-like centromeric protein A
    • Complex: Histone H4
      • Protein or peptide: Histone H4
    • Complex: Histone H2A type 1-B/E
      • Protein or peptide: Histone H2A type 1-B/E
    • Complex: Histone H2B type 2-E
      • Protein or peptide: Histone H2B type 2-E
    • Complex: DNA
      • DNA: DNA (145-mer)
      • DNA: DNA (145-mer)
    • Complex: CENP-C
      • Protein or peptide: CENP-C
    • Complex: Maltodextrin-binding protein,Centromere protein N
      • Protein or peptide: Maltodextrin-binding protein,Centromere protein N

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Supramolecule #1: CENP-A nucleosome in complex with CENP-C peptide and CNEP-N N-ter...

SupramoleculeName: CENP-A nucleosome in complex with CENP-C peptide and CNEP-N N-terminal domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Ectocarpus siliculosus (eukaryote)
Molecular weightTheoretical: 330 KDa

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Supramolecule #2: Histone H3,Histone H3-like centromeric protein A

SupramoleculeName: Histone H3,Histone H3-like centromeric protein A / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Histone H4

SupramoleculeName: Histone H4 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: Histone H2A type 1-B/E

SupramoleculeName: Histone H2A type 1-B/E / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #5: Histone H2B type 2-E

SupramoleculeName: Histone H2B type 2-E / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Escherichia coli (strain B / BL21-DE3) (bacteria)

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Supramolecule #6: DNA

SupramoleculeName: DNA / type: complex / ID: 6 / Parent: 1 / Macromolecule list: #5-#6

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Supramolecule #7: CENP-C

SupramoleculeName: CENP-C / type: complex / ID: 7 / Parent: 1 / Macromolecule list: #7

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Supramolecule #8: Maltodextrin-binding protein,Centromere protein N

SupramoleculeName: Maltodextrin-binding protein,Centromere protein N / type: complex / ID: 8 / Parent: 1 / Macromolecule list: #8

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Macromolecule #1: Histone H3,Histone H3-like centromeric protein A

MacromoleculeName: Histone H3,Histone H3-like centromeric protein A / type: protein_or_peptide / ID: 1
Details: chimeric protein, chicken histone H3 (aa 1-64)-chicken CENP-A (aa 52-131)
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 16.372217 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GSHMARTKQT ARKSTGGKAP RKQLATKAAR KSAPATGGVK KPHRYRPGTV ALREIRRYQK STELLIRRQP FARVVREICL LFTRGVDYR WQAMALLALQ EAAEAFLVHL LEDAYLCSLH ARRVTLYPKD LQLARRLRGL QGEGF

UniProtKB: Histone H3-like centromeric protein A

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Macromolecule #2: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.676703 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSHMSGRGKG GKGLGKGGAK RHRKVLRDNI QGITKPAIRR LARRGGVKRI SGLIYEETRG VLKVFLENVI RDAVTYTEHA KRKTVTAMD VVYALKRQGR TLYGFGG

UniProtKB: Histone H4

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Macromolecule #3: Histone H2A type 1-B/E

MacromoleculeName: Histone H2A type 1-B/E / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.447825 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSHMSGRGKQ GGKARAKAKT RSSRAGLQFP VGRVHRLLRK GNYSERVGAG APVYLAAVLE YLTAEILELA GNAARDNKKT RIIPRHLQL AIRNDEELNK LLGRVTIAQG GVLPNIQAVL LPKKTESHHK AKGK

UniProtKB: Histone H2A type 1-B/E

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Macromolecule #4: Histone H2B type 2-E

MacromoleculeName: Histone H2B type 2-E / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.233518 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSHMPEPAKS APAPKKGSKK AVTKAQKKDG KKRKRSRKES YSIYVYKVLK QVHPDTGISS KAMGIMNSFV NDIFERIAGE ASRLAHYNK RSTITSREIQ TAVRLLLPGE LAKHAVSEGT KAVTKYTSSK

UniProtKB: Histone H2B type 2-E

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Macromolecule #7: CENP-C

MacromoleculeName: CENP-C / type: protein_or_peptide / ID: 7 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 4.93284 KDa
SequenceString:
QKIVLPSNTP NVRRTKRIRL KPLEYWRGER VTYTLKPSGR L

UniProtKB: CENP-C

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Macromolecule #8: Maltodextrin-binding protein,Centromere protein N

MacromoleculeName: Maltodextrin-binding protein,Centromere protein N / type: protein_or_peptide / ID: 8
Details: The fusion protein of N-terminal MBP, N-terminal domain of chicken CENP-N and C-terminal histidine tags
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 71.519625 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKIEEGKLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ VAATGDGPDI IFWAHDRFGG YAQSGLLAEI TPDKAFQDK LYPFTWDAVR YNGKLIAYPI AVEALSLIYN KDLLPNPPKT WEEIPALDKE LKAKGKSALM FNLQEPYFTW P LIAADGGY ...String:
MKIEEGKLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ VAATGDGPDI IFWAHDRFGG YAQSGLLAEI TPDKAFQDK LYPFTWDAVR YNGKLIAYPI AVEALSLIYN KDLLPNPPKT WEEIPALDKE LKAKGKSALM FNLQEPYFTW P LIAADGGY AFKYENGKYD IKDVGVDNAG AKAGLTFLVD LIKNKHMNAD TDYSIAEAAF NKGETAMTIN GPWAWSNIDT SK VNYGVTV LPTFKGQPSK PFVGVLSAGI NAASPNKELA KEFLENYLLT DEGLEAVNKD KPLGAVALKS YEEELAKDPR IAA TMENAQ KGEIMPNIPQ MSAFWYAVRT AVINAASGRQ TVDEALKDAQ TNSSSNNNNN NNNNNLGIEG RENLYFQGRE NLYF QGENL YFQGRIMDEV IVEYIRRTVL KIPRDEIMAV LQKWGFLSEA QLQTINFRQT KEGISHSVAQ LCEESSADLK QAALL DIIY NHIYPNKRVW SVYHMNKTGE ETDFFDFRDF KKKFRRQIQS ALINVTINFR EYEDNAIWIR IAWGTPYTKP NQYKTS YVV YHSQTPYVFI SASVLRSNLP LLCQAMVVAS NYHDIHEMEL RSHCLNSLKD IVFKRYSQND PHHHHHH

UniProtKB: Centromere protein N

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Macromolecule #5: DNA (145-mer)

MacromoleculeName: DNA (145-mer) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 44.529398 KDa
SequenceString: (DA)(DT)(DC)(DA)(DG)(DA)(DA)(DT)(DC)(DC) (DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA)(DG) (DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA) (DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA)(DG) (DA) (DC)(DA)(DG)(DC)(DT)(DC) ...String:
(DA)(DT)(DC)(DA)(DG)(DA)(DA)(DT)(DC)(DC) (DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA)(DG) (DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA) (DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA)(DG) (DA) (DC)(DA)(DG)(DC)(DT)(DC)(DT)(DA) (DG)(DC)(DA)(DC)(DC)(DG)(DC)(DT)(DT)(DA) (DA)(DA) (DC)(DG)(DC)(DA)(DC)(DG)(DT) (DA)(DC)(DG)(DC)(DG)(DC)(DT)(DG)(DT)(DC) (DC)(DC)(DC) (DC)(DG)(DC)(DG)(DT)(DT) (DT)(DT)(DA)(DA)(DC)(DC)(DG)(DC)(DC)(DA) (DA)(DG)(DG)(DG) (DG)(DA)(DT)(DT)(DA) (DC)(DT)(DC)(DC)(DC)(DT)(DA)(DG)(DT)(DC) (DT)(DC)(DC)(DA)(DG) (DG)(DC)(DA)(DC) (DG)(DA)(DG)(DT)(DC)(DA)(DG)(DA)(DT)(DA) (DT)(DA)(DT)(DA)(DC)(DA) (DT)(DC)(DG) (DA)(DT)

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Macromolecule #6: DNA (145-mer)

MacromoleculeName: DNA (145-mer) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 44.982648 KDa
SequenceString: (DA)(DT)(DC)(DG)(DA)(DT)(DG)(DT)(DA)(DT) (DA)(DT)(DA)(DT)(DC)(DT)(DG)(DA)(DC)(DT) (DC)(DG)(DT)(DG)(DC)(DC)(DT)(DG)(DG) (DA)(DG)(DA)(DC)(DT)(DA)(DG)(DG)(DG)(DA) (DG) (DT)(DA)(DA)(DT)(DC)(DC) ...String:
(DA)(DT)(DC)(DG)(DA)(DT)(DG)(DT)(DA)(DT) (DA)(DT)(DA)(DT)(DC)(DT)(DG)(DA)(DC)(DT) (DC)(DG)(DT)(DG)(DC)(DC)(DT)(DG)(DG) (DA)(DG)(DA)(DC)(DT)(DA)(DG)(DG)(DG)(DA) (DG) (DT)(DA)(DA)(DT)(DC)(DC)(DC)(DC) (DT)(DT)(DG)(DG)(DC)(DG)(DG)(DT)(DT)(DA) (DA)(DA) (DA)(DC)(DG)(DC)(DG)(DG)(DG) (DG)(DG)(DA)(DC)(DA)(DG)(DC)(DG)(DC)(DG) (DT)(DA)(DC) (DG)(DT)(DG)(DC)(DG)(DT) (DT)(DT)(DA)(DA)(DG)(DC)(DG)(DG)(DT)(DG) (DC)(DT)(DA)(DG) (DA)(DG)(DC)(DT)(DG) (DT)(DC)(DT)(DA)(DC)(DG)(DA)(DC)(DC)(DA) (DA)(DT)(DT)(DG)(DA) (DG)(DC)(DG)(DG) (DC)(DC)(DT)(DC)(DG)(DG)(DC)(DA)(DC)(DC) (DG)(DG)(DG)(DA)(DT)(DT) (DC)(DT)(DG) (DA)(DT)

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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

Concentration1.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
100.0 mMNaClsodium chloride
2.0 mMC4H10O2S2DTT
1.0 mMC10H16N2O8EDTA
0.1 %C32H58N2O7SCHAPS
GridModel: Quantifoil R0.6/1 / Material: MOLYBDENUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 30 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: AIR / Details: Both side
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL CRYO ARM 200
TemperatureMin: 80.0 K / Max: 80.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 5 / Number real images: 5346 / Average exposure time: 10.0 sec. / Average electron dose: 1.2 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 150.0 µm / Calibrated defocus max: -7.0 µm / Calibrated defocus min: -0.5 µm / Calibrated magnification: 45454 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.4 mm / Nominal defocus max: -3.5 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: JEOL CRYOSPECPORTER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 421635
Startup modelType of model: EMDB MAP
EMDB ID:

8140

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.08) / Number images used: 118294
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.08)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.08)
Final 3D classificationNumber classes: 5 / Avg.num./class: 80000 / Software - Name: RELION (ver. 3.08)
FSC plot (resolution estimation)

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