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- EMDB-24667: Cryo-EM structure of Kip3 (AMPPNP) bound to GMPCPP-Stabilized Mic... -

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Basic information

Entry
Database: EMDB / ID: EMD-24667
TitleCryo-EM structure of Kip3 (AMPPNP) bound to GMPCPP-Stabilized Microtubules
Map dataCryo-EM structure of Kip3 (AMPPNP) bound to GMPCPP-Stabilized Microtubules
Sample
  • Complex: Cryo-EM structure of Kip3 (AMPPNP) bound to GMPCPP-Stabilized Microtubules
    • Complex: GMPCPP-stabilized microtubules
      • Protein or peptide: Tubulin alpha-1B chain
      • Protein or peptide: Tubulin beta chain
    • Complex: yeast kinesin-8/ Kip3
      • Protein or peptide: kinesin-8/ Kip3
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Keywordskinesin-8 / microtubules / complex / MOTOR PROTEIN
Function / homology
Function and homology information


Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling ...Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / Recruitment of NuMA to mitotic centrosomes / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / COPI-independent Golgi-to-ER retrograde traffic / MHC class II antigen presentation / COPI-mediated anterograde transport / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / microtubule / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain ...Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
Tubulin beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesSus scrofa (pig) / Saccharomyces cerevisiae (brewer's yeast)
Methodhelical reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsHernandez-Lopez RA / Leschziner AE
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Biorxiv / Year: 2024
Title: Multimodal tubulin binding by the yeast kinesin-8, Kip3, underlies its motility and depolymerization
Authors: Arellano-Santoyo H / Hernandez-Lopez RA / Stokasimov E / Wang RY-R / Pellman D / Lesczhiner AE
History
DepositionAug 11, 2021-
Header (metadata) releaseAug 17, 2022-
Map releaseAug 17, 2022-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24667.png

Downloads & links

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Map

FileDownload / File: emd_24667.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of Kip3 (AMPPNP) bound to GMPCPP-Stabilized Microtubules
Voxel sizeX=Y=Z: 0.98 Å
Density
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-10.953051 - 14.474532999999999
Average (Standard dev.)0.0077742627 (±0.36639103)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 294.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Cryo-EM structure of Kip3 (AMPPNP) bound to GMPCPP-Stabilized Mic...

EntireName: Cryo-EM structure of Kip3 (AMPPNP) bound to GMPCPP-Stabilized Microtubules
Components
  • Complex: Cryo-EM structure of Kip3 (AMPPNP) bound to GMPCPP-Stabilized Microtubules
    • Complex: GMPCPP-stabilized microtubules
      • Protein or peptide: Tubulin alpha-1B chain
      • Protein or peptide: Tubulin beta chain
    • Complex: yeast kinesin-8/ Kip3
      • Protein or peptide: kinesin-8/ Kip3
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: Cryo-EM structure of Kip3 (AMPPNP) bound to GMPCPP-Stabilized Mic...

SupramoleculeName: Cryo-EM structure of Kip3 (AMPPNP) bound to GMPCPP-Stabilized Microtubules
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: GMPCPP-stabilized microtubules

SupramoleculeName: GMPCPP-stabilized microtubules / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Sus scrofa (pig)

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Supramolecule #3: yeast kinesin-8/ Kip3

SupramoleculeName: yeast kinesin-8/ Kip3 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 1 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 50.204445 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLISQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRSIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDSVEGEGE EEGEEY

UniProtKB: Tubulin alpha-1B chain

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Macromolecule #2: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 49.90777 KDa
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATADEQGE FEEEGEEDEA

UniProtKB: Tubulin beta chain

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Macromolecule #3: kinesin-8/ Kip3

MacromoleculeName: kinesin-8/ Kip3 / type: protein_or_peptide / ID: 3 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 39.900332 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MNVPETRQSS IVVAIRVRPF TSMEKTRLVI RKIVDCVDDR MLIFDPADRN SNATNKFSSQ RRRHGGEIKF VFDKLFDETS SQARVYKET TSPLLDSVLD GFNSTVFAYG ATGCGKTYTV SGTPSQPGII FLAMEELFNK ITDLKDEKDF EISLSYLEIY N ERIRDLLK ...String:
MNVPETRQSS IVVAIRVRPF TSMEKTRLVI RKIVDCVDDR MLIFDPADRN SNATNKFSSQ RRRHGGEIKF VFDKLFDETS SQARVYKET TSPLLDSVLD GFNSTVFAYG ATGCGKTYTV SGTPSQPGII FLAMEELFNK ITDLKDEKDF EISLSYLEIY N ERIRDLLK PETPSKRLVI REDTQNHIKV ANLSYHHPNT VEDVMDLVVQ GNINRTTSPT EANEVSSRSH AVLQIHIMQT NK LVDLTSQ HTFATLSIID LAGSERAAAT RNRGIRLHEG ANINRSLLAL GNCINALCLN DGSRSCHIPY RDSKLTRLLK FSL GGNCKT VMIVCISPSS SHYDETLNTL KYANRAKEI

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 9 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 27 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / type: ligand / ID: 6 / Number of copies: 9 / Formula: G2P
Molecular weightTheoretical: 521.208 Da
Chemical component information

ChemComp-G2P:
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GMP-CPP, energy-carrying molecule analogue*YM

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Macromolecule #7: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 7 / Number of copies: 9 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 6.8
Details: BRB80 buffer (80 mM PIPES-KOH, pH 6.8; 1 mM MgCl2, 1 mM EGTA, 1 mM DTT)
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 22 K / Instrument: FEI VITROBOT MARK IV
DetailsHighly purified, glycerol-free tubulin (Cytoskeleton, Inc.) was resuspended in BRB80 buffer (80 mM PIPES-KOH, pH 6.8, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT) to a concentration of 10 mg/mL. GMPCPP microtubules were polymerized using GMPCPP seeds (20 uM tubulin, 1mM GMPCPP, 1mM MgCl2, 1mM DTT in BRB80). A 1 uL aliquot of seeds was transferred to a 37 C water bath. After 30 minutes, 40 uL of elongation mix (2 uM tubulin, 0.5 mM GMPCPP, 0.5 mM MgCl2, 1 mM DTT in BRB80) were added and the mixture was then incubated for additional 4.5 - 5 hours at 37 C. Purified Kip3 438 protein was buffer exchanged to cryoEM buffer (50 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT supplemented with 2mM AMPPNP) and desalted using a ZEBA spin desalting column. The protein was recovered by centrifugation at 15,000 rcf for 2 min. A final spin at 30,000 x g in a TLA 100 rotor (Beckman) for 10 min at 4 C was carried out to remove big aggregates.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
DetailsImages were recorded using a semi-automated acquisition program Serial EM with a defocus range from 1.5 to 3.5 um.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 643 / Average exposure time: 5.0 sec. / Average electron dose: 40.0 e/Å2
Details: Final accumulated electron doses were 40 electrons/A2. Images were collected in super-resolution mode. The total exposure time was 5 seconds, fractionated into 20 subframes, each with an exposure time of 0.25 s.
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 29090 / Software - Name: Appion
Details: Inspection, defocus estimation, microtubule picking, and stack creation were performed within the Appion processing environment (Lander et al., 2009). Images were selected for processing on ...Details: Inspection, defocus estimation, microtubule picking, and stack creation were performed within the Appion processing environment (Lander et al., 2009). Images were selected for processing on the basis of high decoration, straight MTs, and the absence of crystalline ice.
Startup modelType of model: INSILICO MODEL
Final angle assignmentType: NOT APPLICABLE / Software - Name: FREALIGN
Final reconstructionApplied symmetry - Helical parameters - Δz: 8.9 Å
Applied symmetry - Helical parameters - Δ&Phi: -25.76 °
Applied symmetry - Helical parameters - Axial symmetry: C14 (14 fold cyclic)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN / Number images used: 21788

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Atomic model buiding 1

Initial model
PDB IDChain

3jat

chain_id: A, source_name: PDB, initial_model_type: experimental model

3jat

chain_id: B, source_name: PDB, initial_model_type: experimental model

4frz

chain_id: K, source_name: PDB, initial_model_type: experimental model
DetailsMultiple rounds of refinement were carried out against one half map (training map), and the other half map (validation map) was used to monitor overfitting based on the procedure described in Wang et al. elife, 2016. It is to note that the molecular interactions of ligand-protein were restrained to the initial poses adapted from the high-resolution structures during structure refinement.
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 100
Target criteria: Overall correlation of the residues to the map
Output model

PDB-7rs6:
Cryo-EM structure of Kip3 (AMPPNP) bound to GMPCPP-Stabilized Microtubules

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