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- EMDB-8266: Near-atomic cryo-EM structure of PRC1 bound to the microtubule -

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Basic information

Entry
Database: EMDB / ID: EMD-8266
TitleNear-atomic cryo-EM structure of PRC1 bound to the microtubule
Map dataNear-atomic cryo-EM structure of PRC1 bound to the microtubule
Sample
  • Complex: Ternary complex of PRC1 fragment (PRC1-SC), alpha-tubulin, and beta-tubulin.
    • Protein or peptide: Tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Protein regulator of cytokinesis 1
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: Peloruside A
Keywordscytoskeleton / mitosis / microtubule / microtubule-associated protein / STRUCTURAL PROTEIN
Function / homology
Function and homology information


contractile ring / mitotic spindle midzone assembly / mitotic spindle elongation / mitotic spindle midzone / 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 ...contractile ring / mitotic spindle midzone assembly / mitotic spindle elongation / mitotic spindle midzone / 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 / RHO GTPases activate CIT / 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 / intercellular bridge / kinesin binding / regulation of cytokinesis / spindle microtubule / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / spindle / microtubule cytoskeleton organization / spindle pole / microtubule cytoskeleton / mitotic cell cycle / chromosome / midbody / microtubule binding / microtubule / cell division / GTPase activity / positive regulation of cell population proliferation / GTP binding / protein kinase binding / nucleoplasm / identical protein binding / metal ion binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Microtubule-associated protein, MAP65/Ase1/PRC1 / Microtubule associated protein (MAP65/ASE1 family) / 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 ...Microtubule-associated protein, MAP65/Ase1/PRC1 / Microtubule associated protein (MAP65/ASE1 family) / 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
Protein regulator of cytokinesis 1 / Tubulin beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesSus scrofa (pig) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKellogg EH / Howes S
Funding support United States, Spain, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM051487 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM65933 United States
Ministerio de Economia y CompetitividadBFU2013-44306-P Spain
CitationJournal: Proc Natl Acad Sci U S A / Year: 2016
Title: Near-atomic cryo-EM structure of PRC1 bound to the microtubule.
Authors: Elizabeth H Kellogg / Stuart Howes / Shih-Chieh Ti / Erney Ramírez-Aportela / Tarun M Kapoor / Pablo Chacón / Eva Nogales /
Abstract: Proteins that associate with microtubules (MTs) are crucial to generate MT arrays and establish different cellular architectures. One example is PRC1 (protein regulator of cytokinesis 1), which cross- ...Proteins that associate with microtubules (MTs) are crucial to generate MT arrays and establish different cellular architectures. One example is PRC1 (protein regulator of cytokinesis 1), which cross-links antiparallel MTs and is essential for the completion of mitosis and cytokinesis. Here we describe a 4-Å-resolution cryo-EM structure of monomeric PRC1 bound to MTs. Residues in the spectrin domain of PRC1 contacting the MT are highly conserved and interact with the same pocket recognized by kinesin. We additionally found that PRC1 promotes MT assembly even in the presence of the MT stabilizer taxol. Interestingly, the angle of the spectrin domain on the MT surface corresponds to the previously observed cross-bridge angle between MTs cross-linked by full-length, dimeric PRC1. This finding, together with molecular dynamic simulations describing the intrinsic flexibility of PRC1, suggests that the MT-spectrin domain interface determines the geometry of the MT arrays cross-linked by PRC1.
History
DepositionJul 21, 2016-
Header (metadata) releaseAug 3, 2016-
Map releaseAug 3, 2016-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.27
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 4.27
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5kmg
  • Surface level: 4.27
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5kmg
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8266.png

Downloads & links

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Map

FileDownload / File: emd_8266.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNear-atomic cryo-EM structure of PRC1 bound to the microtubule
Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 4.27 / Movie #1: 4.27
Minimum - Maximum-16.888908000000001 - 31.584765999999998
Average (Standard dev.)0.0872013 (±1.2133477)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 675.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z675.840675.840675.840
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-16.88931.5850.087

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Supplemental data

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Sample components

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Entire : Ternary complex of PRC1 fragment (PRC1-SC), alpha-tubulin, and be...

EntireName: Ternary complex of PRC1 fragment (PRC1-SC), alpha-tubulin, and beta-tubulin.
Components
  • Complex: Ternary complex of PRC1 fragment (PRC1-SC), alpha-tubulin, and beta-tubulin.
    • Protein or peptide: Tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Protein regulator of cytokinesis 1
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: Peloruside A

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Supramolecule #1: Ternary complex of PRC1 fragment (PRC1-SC), alpha-tubulin, and be...

SupramoleculeName: Ternary complex of PRC1 fragment (PRC1-SC), alpha-tubulin, and beta-tubulin.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: The PRC1 fragment referred to as PRC1-SC contains the spectrin domain and the unstructured C-terminal domain, spanning (337-620)

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Macromolecule #1: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig) / Tissue: brain
Molecular weightTheoretical: 49.095438 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 GVDSVE

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: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig) / Tissue: brain
Molecular weightTheoretical: 48.299293 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 QDATAD

UniProtKB: Tubulin beta chain

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Macromolecule #3: Protein regulator of cytokinesis 1

MacromoleculeName: Protein regulator of cytokinesis 1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.69394 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GAAALKNYYE VHKELFEGVQ KWEETWRLFL EFERKASDPN RFTNRGGNLL KEEKQRAKLQ KMLPKLEEEL KARIELWEQE HSKAFMVNG QKFMEYVAEQ WEMHRLEKER AKQERQLKNK KQTETEMLY

UniProtKB: Protein regulator of cytokinesis 1

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / 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: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM / Guanosine diphosphate

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Macromolecule #7: Peloruside A

MacromoleculeName: Peloruside A / type: ligand / ID: 7 / Number of copies: 1 / Formula: POU
Molecular weightTheoretical: 548.663 Da
Chemical component information

ChemComp-POU:
Peloruside A

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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

Concentration0.4 mg/mL
BufferpH: 6.8
Component:
ConcentrationFormulaName
80.0 mMC8H18N2O6S2PIPES
1.0 mMMgCl2magnesium chloride
1.0 mMC14H24N2O10EGTA
1.0 mMC4H10O2S2DTT
0.05 % voloctylphenoxypolyethoxyethanolNP-40
GridModel: protochips CF-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 4 seconds, blot force 10.

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Electron microscopy

MicroscopeFEI TITAN
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 3.8000000000000003 µm / Calibrated defocus min: 1.1 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 27500
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
TemperatureMin: 93.0 K / Max: 93.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-20 / Number grids imaged: 1 / Number real images: 312 / Average exposure time: 0.3 sec. / Average electron dose: 8.0 e/Å2

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Image processing

Segment selectionNumber selected: 27275
Details: manually selected microtubule filaments using manualpicker.py
Startup modelType of model: OTHER
Details: low-pass filtered reference model of microtubules decorated with kinesin
Final angle assignmentType: NOT APPLICABLE / Software - Name: FREALIGN (ver. 9.09)
Final reconstructionApplied symmetry - Helical parameters - Δz: 8.67 Å
Applied symmetry - Helical parameters - Δ&Phi: -25.76 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.09) / Number images used: 17069

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Atomic model buiding 1

Initial model
PDB IDChain

3nrx

chain_id: A, residue_range: 1-130, source_name: PDB, initial_model_type: experimental model

4l6y

chain_id: A, residue_range: 347-450, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: correlation coefficient
Output model

PDB-5kmg:
Near-atomic cryo-EM structure of PRC1 bound to the microtubule

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