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- EMDB-25778: Uncrosslinked VRK1-nucleosome complex -

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Basic information

Entry
Database: EMDB / ID: EMD-25778
TitleUncrosslinked VRK1-nucleosome complex
Map datauncrosslinked VRK1-nucleosome complex
Sample
  • Complex: Structure of VRK1 C-terminal tail bound to nucleosome core particle
    • Complex: Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B type 1-C/E/F/G/I, Serine/threonine-protein kinase VRK1
      • Protein or peptide: Histone H3.2
      • Protein or peptide: Histone H4
      • Protein or peptide: Histone H2A
      • Protein or peptide: Histone H2B
      • Protein or peptide: Vaccinia-related kinase 1
    • Complex: DNA
      • DNA: WIDOM 601 DNA (185-MER)
      • DNA: WIDOM 601 DNA (185-MER)
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsSpangler CJ / Budziszewski GR / McGinty RK
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM133498 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM119999 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)F99CA253730 United States
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Multivalent DNA and nucleosome acidic patch interactions specify VRK1 mitotic localization and activity.
Authors: Gabrielle R Budziszewski / Yani Zhao / Cathy J Spangler / Katarzyna M Kedziora / Michael R Williams / Dalal N Azzam / Aleksandra Skrajna / Yuka Koyama / Andrew P Cesmat / Holly C Simmons / ...Authors: Gabrielle R Budziszewski / Yani Zhao / Cathy J Spangler / Katarzyna M Kedziora / Michael R Williams / Dalal N Azzam / Aleksandra Skrajna / Yuka Koyama / Andrew P Cesmat / Holly C Simmons / Eyla C Arteaga / Joshua D Strauss / Dmitri Kireev / Robert K McGinty /
Abstract: A key role of chromatin kinases is to phosphorylate histone tails during mitosis to spatiotemporally regulate cell division. Vaccinia-related kinase 1 (VRK1) is a serine-threonine kinase that ...A key role of chromatin kinases is to phosphorylate histone tails during mitosis to spatiotemporally regulate cell division. Vaccinia-related kinase 1 (VRK1) is a serine-threonine kinase that phosphorylates histone H3 threonine 3 (H3T3) along with other chromatin-based targets. While structural studies have defined how several classes of histone-modifying enzymes bind to and function on nucleosomes, the mechanism of chromatin engagement by kinases is largely unclear. Here, we paired cryo-electron microscopy with biochemical and cellular assays to demonstrate that VRK1 interacts with both linker DNA and the nucleosome acidic patch to phosphorylate H3T3. Acidic patch binding by VRK1 is mediated by an arginine-rich flexible C-terminal tail. Homozygous missense and nonsense mutations of this acidic patch recognition motif in VRK1 are causative in rare adult-onset distal spinal muscular atrophy. We show that these VRK1 mutations interfere with nucleosome acidic patch binding, leading to mislocalization of VRK1 during mitosis, thus providing a potential new molecular mechanism for pathogenesis.
History
DepositionDec 21, 2021-
Header (metadata) releaseMay 4, 2022-
Map releaseMay 4, 2022-
UpdateMay 18, 2022-
Current statusMay 18, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25778.png

Downloads & links

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Map

FileDownload / File: emd_25778.map.gz / Format: CCP4 / Size: 137.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationuncrosslinked VRK1-nucleosome complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 330 pix.
= 300.3 Å		0.91 Å/pix.
x 330 pix.
= 300.3 Å		0.91 Å/pix.
x 330 pix.
= 300.3 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.91 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.017
Minimum - Maximum-0.03773744 - 0.063925155
Average (Standard dev.)0.000128736 (±0.0015598391)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions330330330
Spacing330330330
CellA=B=C: 300.30002 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Structure of VRK1 C-terminal tail bound to nucleosome core particle

EntireName: Structure of VRK1 C-terminal tail bound to nucleosome core particle
Components
  • Complex: Structure of VRK1 C-terminal tail bound to nucleosome core particle
    • Complex: Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B type 1-C/E/F/G/I, Serine/threonine-protein kinase VRK1
      • Protein or peptide: Histone H3.2
      • Protein or peptide: Histone H4
      • Protein or peptide: Histone H2A
      • Protein or peptide: Histone H2B
      • Protein or peptide: Vaccinia-related kinase 1
    • Complex: DNA
      • DNA: WIDOM 601 DNA (185-MER)
      • DNA: WIDOM 601 DNA (185-MER)

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Supramolecule #1: Structure of VRK1 C-terminal tail bound to nucleosome core particle

SupramoleculeName: Structure of VRK1 C-terminal tail bound to nucleosome core particle
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#7

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Supramolecule #2: Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B type 1-...

SupramoleculeName: Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B type 1-C/E/F/G/I, Serine/threonine-protein kinase VRK1
type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1-#4, #7
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #5-#6
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Histone H3.2

MacromoleculeName: Histone H3.2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
ARTKQTARKS TGGKAPRKQL ATKAARKSAP ATGGVKKPHR YRPGTVALRE IRRYQKSTEL LIRKLPFQRL VREIAQDFKT DLRFQSSAVM ALQEASEAYL VGLFEDTNLC AIHAKRVTIM PKDIQLARRI RGERA

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Macromolecule #2: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKGGKG LGKGGAKRHR KVLRDNIQGI TKPAIRRLAR RGGVKRISGL IYEETRGVLK VFLENVIRDA VTYTEHAKRK TVTAMDVVYA LKRQGRTLYG FGG

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Macromolecule #3: Histone H2A

MacromoleculeName: Histone H2A / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGRGKQGGKA RAKAKTRSSR AGLQFPVGRV HRLLRKGNYA ERVGAGAPVY LAAVLEYLTA EILELAGNAA RDNKKTRIIP RHLQLAIRND EELNKLLGKV TIAQGGVLPN IQAVLLPKKT ESHHKAKGK

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Macromolecule #4: Histone H2B

MacromoleculeName: Histone H2B / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
PEPAKSAPAP KKGSKKAVTK AQKKDGKKRK RSRKESYSVY VYKVLKQVHP DTGISSKAMG IMNSFVNDIF ERIAGEASRL AHYNKRSTIT SREIQTAVRL LLPGELAKHA VSEGTKAVTK YTSSK

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Macromolecule #7: Vaccinia-related kinase 1

MacromoleculeName: Vaccinia-related kinase 1 / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSMPRVKAAQ AGRQSSAKRH LAEQFAVGEI ITDMAKKEWK VGLPIGQGGF GCIYLADMNS SESVGSDAPC VVKVEPSDNG PLFTELKFYQ RAAKPEQIQK WIRTRKLKYL GVPKYWGSGL HDKNGKSYRF MIMDRFGSDL QKIYEANAKR FSRKTVLQLS LRILDILEYI ...String:
GSMPRVKAAQ AGRQSSAKRH LAEQFAVGEI ITDMAKKEWK VGLPIGQGGF GCIYLADMNS SESVGSDAPC VVKVEPSDNG PLFTELKFYQ RAAKPEQIQK WIRTRKLKYL GVPKYWGSGL HDKNGKSYRF MIMDRFGSDL QKIYEANAKR FSRKTVLQLS LRILDILEYI HEHEYVHGDI KASNLLLNYK NPDQVYLVDY GLAYRYCPEG VHKEYKEDPK RCHDGTIEFT SIDAHNGVAP SRRGDLEILG YCMIQWLTGH LPWEDNLKDP KYVRDSKIRY RENIASLMDK CFPEKNKPGE IAKYMETVKL LDYTEKPLYE NLRDILLQGL KAIGSKDDGK LDLSVVENGG LKAKTITKKR KKEIEESKEP GVEDTEWSNT QTEEAIQTRS RTRKRVQK

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Macromolecule #5: WIDOM 601 DNA (185-MER)

MacromoleculeName: WIDOM 601 DNA (185-MER) / type: dna / ID: 5 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
ATCGCTGTTC AATACATGCA CAGGATGTAT ATATCTGACA CGTGCCTGGA GACTAGGGAG TAATCCCCTT GGCGGTTAAA ACGCGGGGGA CAGCGCGTAC GTGCGTTTAA GCGGTGCTAG AGCTGTCTAC GACCAATTGA GCGGCCTCGG CACCGGGATT CTCCAGGGCG GCCGCGTATA GGGAT

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Macromolecule #6: WIDOM 601 DNA (185-MER)

MacromoleculeName: WIDOM 601 DNA (185-MER) / type: dna / ID: 6 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
ATCCCTATAC GCGGCCGCCC TGGAGAATCC CGGTGCCGAG GCCGCTCAAT TGGTCGTAGA CAGCTCTAGC ACCGCTTAAA CGCACGTACG CGCTGTCCCC CGCGTTTTAA CCGCCAAGGG GATTACTCCC TAGTCTCCAG GCACGTGTCA GATATATACA TCCTGTGCAT GTATTGAACA GCGAT

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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: 7.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 3072 / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6esf


Details: low pass filtered at 50 angstroms
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 53704
FSC plot (resolution estimation)

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