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

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Basic information

Entry
Database: PDB / ID: 5kmg
TitleNear-atomic cryo-EM structure of PRC1 bound to the microtubule
Components
  • Protein regulator of cytokinesis 1
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / cytoskeleton / mitosis / microtubule / microtubule-associated 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
Methane Monooxygenase Hydroxylase; Chain G, domain 1 - #1520 / Microtubule-associated protein, MAP65/Ase1/PRC1 / Microtubule associated protein (MAP65/ASE1 family) / Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site ...Methane Monooxygenase Hydroxylase; Chain G, domain 1 - #1520 / Microtubule-associated protein, MAP65/Ase1/PRC1 / Microtubule associated protein (MAP65/ASE1 family) / Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / 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 / Methane Monooxygenase Hydroxylase; Chain G, domain 1 / Helix Hairpins / Up-down Bundle / Rossmann fold / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Peloruside A / Protein regulator of cytokinesis 1 / Tubulin beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKellogg, E.H. / Howes, S. / Ti, S.-C. / Ramirez-Aportela, E. / Kapoor, T.M. / Chacon, P. / Nogales, E.
Funding support United States, Spain, 3items
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
DepositionJun 27, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 3, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 24, 2016Group: Database references
Revision 1.2Sep 7, 2016Group: Database references
Revision 1.3Sep 27, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.4Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Mar 6, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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  • Biological unit as representative helical assembly
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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Tubulin alpha-1B chain
B: Tubulin beta chain
P: Protein regulator of cytokinesis 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)114,6287
Polymers113,0893
Non-polymers1,5394
Water0
1
A: Tubulin alpha-1B chain
B: Tubulin beta chain
P: Protein regulator of cytokinesis 1
hetero molecules
x 14


Theoretical massNumber of molelcules
Total (without water)1,604,79298
Polymers1,583,24142
Non-polymers21,55156
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
helical symmetry operation13
2


  • Idetical with deposited unit in distinct coordinate
  • helical asymmetric unit
TypeNameSymmetry operationNumber
helical symmetry operation1
3


  • Idetical with deposited unit
  • helical asymmetric unit, std helical frame
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 14 / Rise per n subunits: 8.57 Å / Rotation per n subunits: -25.76 °)

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Components

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Protein , 3 types, 3 molecules ABP

#1: Protein Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 49095.438 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: brain / References: UniProt: Q2XVP4
#2: Protein Tubulin beta chain / Beta-tubulin


Mass: 48299.293 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: brain / References: UniProt: P02554
#3: Protein Protein regulator of cytokinesis 1


Mass: 15693.940 Da / Num. of mol.: 1 / Fragment: UNP residues 341-464
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRC1 / Production host: Escherichia coli (E. coli) / References: UniProt: O43663

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Non-polymers , 4 types, 4 molecules

#4: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#7: Chemical ChemComp-POU / Peloruside A / (1R,3R,4S,7S,9S,11S,13R,14R,15R)-4,11,13,14-tetrahydroxy-7-[(2Z,4R)-4-(hydroxymethyl)hex-2-en-2-yl]-3,9,15-trimethoxy-12,12-dimethyl-6,17-dioxabicyclo[11.3.1]heptadecan-5-one


Mass: 548.663 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H48O11

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Ternary complex of PRC1 fragment (PRC1-SC), alpha-tubulin, and beta-tubulin.
Type: COMPLEX
Details: The PRC1 fragment referred to as PRC1-SC contains the spectrin domain and the unstructured C-terminal domain, spanning (337-620)
Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Buffer solutionpH: 6.8
Buffer component
IDConc.NameFormulaBuffer-ID
180 mMPIPESC8H18N2O6S21
21 mMmagnesium chlorideMgCl21
31 mMEGTAC14H24N2O101
41 mMDTTC4H10O2S21
50.05 % volNP-40octylphenoxypolyethoxyethanol1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: protochips CF-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: blot for 4 seconds, blot force 10

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 27500 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1100 nm / Calibrated defocus max: 3800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Temperature (max): 93 K / Temperature (min): 93 K
Image recordingAverage exposure time: 0.3 sec. / Electron dose: 8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 312
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 20 / Used frames/image: 1-20

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Processing

EM software
IDNameVersionCategoryDetails
2Leginon3.2image acquisitionleginon was used to collect images
3Appion3.2image acquisitionappion was used to store and do preliminary image processing
5CTFFIND4CTF correction
8UCSF Chimera1.1model fitting
10I-TASSER4.4model refinement
11iMODFIT1.04model refinement
12Rosetta3.6model refinement
13EMAN22.07initial Euler assignment
14FREALIGN9.09final Euler assignment
16FREALIGN9.093D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -25.76 ° / Axial rise/subunit: 8.67 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 27275
Details: manually selected microtubule filaments using manualpicker.py
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17069 / Algorithm: FOURIER SPACE / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: correlation coefficient
Atomic model building

3D fitting-ID: 1 / Pdb chain-ID: A / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeInitial refinement model-IDPdb chain residue range
13NRX

3nrx

3NRX11-130
24L6Y

4l6y

4L6Y2347-450

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