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- PDB-4nf9: Structure of the Knl1/Nsl1 complex -

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Basic information

Entry
Database: PDB / ID: 4nf9
TitleStructure of the Knl1/Nsl1 complex
Components
  • Kinetochore-associated protein NSL1 homolog
  • Protein CASC5
KeywordsCELL CYCLE / RWD domain
Function / homology
Function and homology information


Knl1/Spc105 complex / positive regulation of meiosis I spindle assembly checkpoint / homologous chromosome orientation in meiotic metaphase I / MIS12/MIND type complex / acrosome assembly / regulation of mitotic cell cycle spindle assembly checkpoint / attachment of spindle microtubules to kinetochore / protein localization to kinetochore / mitotic sister chromatid segregation / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal ...Knl1/Spc105 complex / positive regulation of meiosis I spindle assembly checkpoint / homologous chromosome orientation in meiotic metaphase I / MIS12/MIND type complex / acrosome assembly / regulation of mitotic cell cycle spindle assembly checkpoint / attachment of spindle microtubules to kinetochore / protein localization to kinetochore / mitotic sister chromatid segregation / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Deposition of new CENPA-containing nucleosomes at the centromere / Resolution of Sister Chromatid Cohesion / acrosomal vesicle / RHO GTPases Activate Formins / kinetochore / spindle pole / Separation of Sister Chromatids / nuclear body / nuclear speck / cell division / nucleoplasm / nucleus / cytosol
Similarity search - Function
Knl1, C-terminal RWD domain / Knl1 RWD C-terminal domain / Kinetochore Mis14/Nsl1 / Kinetochore scaffold 1 / KNL1 MELT repeat / Kinetochore protein Mis14 like / MELT motif
Similarity search - Domain/homology
Kinetochore scaffold 1 / Kinetochore-associated protein NSL1 homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.8 Å
AuthorsPetrovic, A. / Mosalaganti, S. / Keller, J. / Mattiuzzo, M. / Overlack, K. / Wohlgemuth, S. / Pasqualato, S. / Raunser, S. / Musacchio, A.
CitationJournal: Mol Cell / Year: 2014
Title: Modular assembly of RWD domains on the Mis12 complex underlies outer kinetochore organization.
Authors: Arsen Petrovic / Shyamal Mosalaganti / Jenny Keller / Marta Mattiuzzo / Katharina Overlack / Veronica Krenn / Anna De Antoni / Sabine Wohlgemuth / Valentina Cecatiello / Sebastiano ...Authors: Arsen Petrovic / Shyamal Mosalaganti / Jenny Keller / Marta Mattiuzzo / Katharina Overlack / Veronica Krenn / Anna De Antoni / Sabine Wohlgemuth / Valentina Cecatiello / Sebastiano Pasqualato / Stefan Raunser / Andrea Musacchio /
Abstract: Faithful chromosome segregation is mandatory for cell and organismal viability. Kinetochores, large protein assemblies embedded in centromeric chromatin, establish a mechanical link between ...Faithful chromosome segregation is mandatory for cell and organismal viability. Kinetochores, large protein assemblies embedded in centromeric chromatin, establish a mechanical link between chromosomes and spindle microtubules. The KMN network, a conserved 10-subunit kinetochore complex, harbors the microtubule-binding interface. RWD domains in the KMN subunits Spc24 and Spc25 mediate kinetochore targeting of the microtubule-binding subunits by interacting with the Mis12 complex, a KMN subcomplex that tethers directly onto the underlying chromatin layer. Here, we show that Knl1, a KMN subunit involved in mitotic checkpoint signaling, also contains RWD domains that bind the Mis12 complex and that mediate kinetochore targeting of Knl1. By reporting the first 3D electron microscopy structure of the KMN network, we provide a comprehensive framework to interpret how interactions of RWD-containing proteins with the Mis12 complex shape KMN network topology. Our observations unveil a regular pattern in the construction of the outer kinetochore.
History
DepositionOct 31, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 19, 2014Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein CASC5
B: Protein CASC5
C: Kinetochore-associated protein NSL1 homolog
D: Kinetochore-associated protein NSL1 homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)58,6095
Polymers58,5744
Non-polymers351
Water82946
1
A: Protein CASC5
C: Kinetochore-associated protein NSL1 homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,3223
Polymers29,2872
Non-polymers351
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1240 Å2
ΔGint-12 kcal/mol
Surface area12550 Å2
MethodPISA
2
B: Protein CASC5
D: Kinetochore-associated protein NSL1 homolog


Theoretical massNumber of molelcules
Total (without water)29,2872
Polymers29,2872
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1110 Å2
ΔGint-7 kcal/mol
Surface area12220 Å2
MethodPISA
Unit cell
Length a, b, c (Å)60.710, 71.630, 177.210
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Protein CASC5 / ALL1-fused gene from chromosome 15q14 protein / AF15q14 / Bub-linking kinetochore protein / Blinkin ...ALL1-fused gene from chromosome 15q14 protein / AF15q14 / Bub-linking kinetochore protein / Blinkin / Cancer susceptibility candidate gene 5 protein / Cancer/testis antigen 29 / CT29 / Kinetochore-null protein 1 / Protein D40/AF15q14


Mass: 26023.043 Da / Num. of mol.: 2 / Fragment: Knl1 C-terminal domain, UNP residues 2117-2337
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CASC5, KIAA1570, KNL1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8NG31
#2: Protein/peptide Kinetochore-associated protein NSL1 homolog


Mass: 3263.921 Da / Num. of mol.: 2 / Fragment: Nsl1 C-terminal tail, UNP residues 256-281 / Source method: obtained synthetically / Details: synthetic peptide of a natural human protein / Source: (synth.) Homo sapiens (human) / References: UniProt: Q96IY1
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 46 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.29 Å3/Da / Density % sol: 62.6 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 16-20 % (v/v) PEG 3350, 220 mM sodium citrate and 100 mM MES (pH 6 or 6.5) and 100 mM Hepes (pH 7), VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1.0006 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL
RadiationScattering type: x-ray
Radiation wavelengthWavelength: 1.0006 Å / Relative weight: 1
ReflectionResolution: 2.8→46.31 Å / Num. obs: 36465 / % possible obs: 99.1 % / Observed criterion σ(F): 2 / Observed criterion σ(I): 2 / Redundancy: 3.93 % / Rmerge(I) obs: 0.087 / Rsym value: 0.101 / Net I/σ(I): 14.09
Reflection shellResolution: 2.8→2.87 Å / % possible all: 99.1

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Processing

Software
NameVersionClassification
XDSdata scaling
MOLREPphasing
REFMAC5.6.0117refinement
XDSdata reduction
XSCALEdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.8→46.31 Å / Cor.coef. Fo:Fc: 0.941 / Cor.coef. Fo:Fc free: 0.904 / SU B: 0.007 / SU ML: 0 / Cross valid method: THROUGHOUT / σ(F): 2.8 / ESU R: 0.263 / ESU R Free: 0.337 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflectionSelection details
Rfree0.26502 983 5 %RANDOM
Rwork0.20693 ---
all0.20989 36465 --
obs0.20989 18673 99.47 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 59.449 Å2
Baniso -1Baniso -2Baniso -3
1--3.75 Å20 Å20 Å2
2--8.9 Å20 Å2
3----5.15 Å2
Refinement stepCycle: LAST / Resolution: 2.8→46.31 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3669 0 1 46 3716
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONr_bond_refined_d0.0154
X-RAY DIFFRACTIONr_angle_refined_deg1.823
LS refinement shellResolution: 2.8→2.872 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.389 65 -
Rwork0.311 1243 -
obs--99.62 %

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