[English] 日本語
Yorodumi
- PDB-5m5l: Pseudo-atomic model of microtubule-bound S. pombe kinesin-5 motor... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 5m5l
TitlePseudo-atomic model of microtubule-bound S. pombe kinesin-5 motor domain in the AMPPNP state (based on cryo-electron microscopy experiment): the N-terminus adopts multiple conformations
Components
  • Kinesin-like protein cut7
  • Tubulin alpha-1D chain
  • Tubulin beta-2B chain
KeywordsMOTOR PROTEIN / microtubule-bound S.pombe kinesin-5 / motor domain / AMPPNP bound state
Function / homology
Function and homology information


mitotic spindle formation (spindle phase one) / mitotic spindle elongation (spindle phase three) / Kinesins / initial mitotic spindle pole body separation / microtubule plus-end directed mitotic chromosome migration / meiotic spindle assembly / meiotic spindle pole / mitotic spindle midzone / mitotic spindle pole body / mitotic spindle midzone assembly ...mitotic spindle formation (spindle phase one) / mitotic spindle elongation (spindle phase three) / Kinesins / initial mitotic spindle pole body separation / microtubule plus-end directed mitotic chromosome migration / meiotic spindle assembly / meiotic spindle pole / mitotic spindle midzone / mitotic spindle pole body / mitotic spindle midzone assembly / spindle elongation / polar microtubule / minus-end-directed microtubule motor activity / plus-end-directed microtubule motor activity / meiotic spindle / positive regulation of axon guidance / microtubule associated complex / microtubule motor activity / mitotic spindle assembly / microtubule-based process / spindle microtubule / kinetochore / structural constituent of cytoskeleton / mitotic spindle / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / nervous system development / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / protein heterodimerization activity / cell division / GTPase activity / GTP binding / ATP hydrolysis activity / ATP binding / nucleus / metal ion binding / cytoplasm
Similarity search - Function
: / : / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain superfamily / Alpha tubulin ...: / : / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain superfamily / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / Kinesin-like protein cut7 / Tubulin alpha-1D chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesSchizosaccharomyces pombe (fission yeast)
Bos taurus (domestic cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.3 Å
AuthorsGoulet, A. / Moores, C.A. / Cross, R.A.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2016
Title: Schizosaccharomyces pombe kinesin-5 switches direction using a steric blocking mechanism.
Authors: Mishan Britto / Adeline Goulet / Syeda Rizvi / Ottilie von Loeffelholz / Carolyn A Moores / Robert A Cross /
Abstract: Cut7, the sole kinesin-5 in Schizosaccharomyces pombe, is essential for mitosis. Like other yeast kinesin-5 motors, Cut7 can reverse its stepping direction, by mechanisms that are currently unclear. ...Cut7, the sole kinesin-5 in Schizosaccharomyces pombe, is essential for mitosis. Like other yeast kinesin-5 motors, Cut7 can reverse its stepping direction, by mechanisms that are currently unclear. Here we show that for full-length Cut7, the key determinant of stepping direction is the degree of motor crowding on the microtubule lattice, with greater crowding converting the motor from minus end-directed to plus end-directed stepping. To explain how high Cut7 occupancy causes this reversal, we postulate a simple proximity sensing mechanism that operates via steric blocking. We propose that the minus end-directed stepping action of Cut7 is selectively inhibited by collisions with neighbors under crowded conditions, whereas its plus end-directed action, being less space-hungry, is not. In support of this idea, we show that the direction of Cut7-driven microtubule sliding can be reversed by crowding it with non-Cut7 proteins. Thus, crowding by either dynein microtubule binding domain or Klp2, a kinesin-14, converts Cut7 from net minus end-directed to net plus end-directed stepping. Biochemical assays confirm that the Cut7 N terminus increases Cut7 occupancy by binding directly to microtubules. Direct observation by cryoEM reveals that this occupancy-enhancing N-terminal domain is partially ordered. Overall, our data point to a steric blocking mechanism for directional reversal through which collisions of Cut7 motor domains with their neighbors inhibit their minus end-directed stepping action, but not their plus end-directed stepping action. Our model can potentially reconcile a number of previous, apparently conflicting, observations and proposals for the reversal mechanism of yeast kinesins-5.
History
DepositionOct 21, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 30, 2016Provider: repository / Type: Initial release
Revision 1.1Dec 14, 2016Group: Database references
Revision 1.2Aug 2, 2017Group: Data collection / Derived calculations ...Data collection / Derived calculations / Experimental preparation / Refinement description
Category: em_3d_fitting / em_image_scans ...em_3d_fitting / em_image_scans / em_sample_support / em_software / pdbx_struct_conn_angle
Item: _em_3d_fitting.target_criteria / _em_sample_support.grid_type ..._em_3d_fitting.target_criteria / _em_sample_support.grid_type / _em_software.name / _em_software.version
Revision 1.3Dec 5, 2018Group: Data collection / Category: em_imaging / Item: _em_imaging.c2_aperture_diameter
Revision 1.4May 8, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-3445
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Tubulin alpha-1D chain
B: Tubulin beta-2B chain
C: Kinesin-like protein cut7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)143,2579
Polymers140,8823
Non-polymers2,3756
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

-
Components

-
Protein , 3 types, 3 molecules ABC

#1: Protein Tubulin alpha-1D chain / Coordinate model: Cα atoms only


Mass: 50236.352 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: Q2HJ86
#2: Protein Tubulin beta-2B chain / Coordinate model: Cα atoms only


Mass: 49907.770 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: Q6B856
#3: Protein Kinesin-like protein cut7 / Cell untimely torn protein 7 / Coordinate model: Cα atoms only


Mass: 40737.527 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Gene: cut7, SPAC25G10.07c / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P24339

-
Non-polymers , 5 types, 6 molecules

#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#6: Chemical ChemComp-GDP / GUANOSINE-5'-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-TA1 / TAXOL


Mass: 853.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM
#8: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM

-
Experimental details

-
Experiment

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

-
Sample preparation

ComponentName: microtubule-bound S. pombe kinesin-5 motor domain in the AMPPNP state
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.14 MDa / Experimental value: NO
Buffer solutionpH: 6.8
Buffer component
IDConc.FormulaBuffer-ID
125 mMPIPES1
230 mMNaCl1
37 mMMgCl21
41 mMEGTA1
55 mMAMPPNP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 294 K

-
Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 68000 X / Nominal defocus min: 700 nm / Cs: 2 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN CT3500 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingElectron dose: 20 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)

-
Processing

EM software
IDNameVersionCategory
1EMANparticle selection
4CTFFIND3CTF correction
5FREALIGNCTF correction
8Flex-EMmodel fitting
9UCSF Chimeramodel fitting
11SPIDERinitial Euler assignment
12FREALIGNfinal Euler assignment
14FREALIGN3D reconstruction
15Flex-EMmodel refinement
16MODELLERmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 9.3 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 144300 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: An initial homology model of S. pombe cut7 kinesin-5 motor domain based on human kinesin-5 structure (PDB 3HQD) was prepared using Modeller. The coordinates of motor bound to an alpha-beta ...Details: An initial homology model of S. pombe cut7 kinesin-5 motor domain based on human kinesin-5 structure (PDB 3HQD) was prepared using Modeller. The coordinates of motor bound to an alpha-beta tubulin dimer (PDB 1JFF) were rigidly fitted into the cryo-EM map using Chimera and refined by flexible fitting using Flex-EM. Structural models of loop5 and loop10 were generated using Modeller. The conformation of the neck-linker and the N-terminus were calculated using a conjugate-gradient energy minimization approach.

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more