[English] 日本語
Yorodumi
- EMDB-2081: Electron cryo-microscopy of microtubule-bound human kinesin-5 mot... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-2081
TitleElectron cryo-microscopy of microtubule-bound human kinesin-5 motor domain in the AMPPNP state (gold cluster in the neck linker V365C).
Map data3D reconstruction of microtubule-bound human kinesin-5 motor domain with AMPPNP bound in the nucleotide-binding site (gold cluster attached to the neck linker, V365C)
Sample
  • Sample: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP bound. A gold cluster is attached to the neck linker (V365C).
  • Protein or peptide: alpha tubulin
  • Protein or peptide: beta tubulin
  • Protein or peptide: Kinesin-5 motor domain
Keywordselectron cryo-microscopy / kinesin / microtubule / mitosis / cancer
Function / homologyKinesin motor domain, conserved site / Alpha tubulin / Beta tubulin, autoregulation binding site
Function and homology information
Biological speciesBos taurus (cattle) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.0 Å
AuthorsGoulet A / Behnke-Parks WM / Sindelar CV / Major J / Rosenfeld SS / Moores CA
CitationJournal: J Biol Chem / Year: 2012
Title: The structural basis of force generation by the mitotic motor kinesin-5.
Authors: Adeline Goulet / William M Behnke-Parks / Charles V Sindelar / Jennifer Major / Steven S Rosenfeld / Carolyn A Moores /
Abstract: Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved ...Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved distinct properties for its spindle-based functions. In this study, we report subnanometer resolution cryoelectron microscopy reconstructions of microtubule-bound human kinesin-5 before and after nucleotide binding and combine this information with studies of the kinetics of nucleotide-induced neck linker and cover strand movement. These studies reveal coupled, nucleotide-dependent conformational changes that explain many of this motor's properties. We find that ATP binding induces a ratchet-like docking of the neck linker and simultaneous, parallel docking of the N-terminal cover strand. Loop L5, the binding site for allosteric inhibitors of kinesin-5, also undergoes a dramatic reorientation when ATP binds, suggesting that it is directly involved in controlling nucleotide binding. Our structures indicate that allosteric inhibitors of human kinesin-5, which are being developed as anti-cancer therapeutics, bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Together, our data reveal the precision with which the molecular mechanism of kinesin-5 motors has evolved for force generation.
History
DepositionApr 19, 2012-
Header (metadata) releaseJul 9, 2012-
Map releaseNov 21, 2012-
UpdateJan 9, 2013-
Current statusJan 9, 2013Processing site: PDBe / Status: Released

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.34
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.34
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2081.jpg

Downloads & links

-
Map

FileDownload / File: emd_2081.map.gz / Format: CCP4 / Size: 348.6 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of microtubule-bound human kinesin-5 motor domain with AMPPNP bound in the nucleotide-binding site (gold cluster attached to the neck linker, V365C)
Voxel sizeX=Y=Z: 2.8 Å
Density
Contour LevelBy AUTHOR: 1.34 / Movie #1: 1.34
Minimum - Maximum-7.33667994 - 6.94212723
Average (Standard dev.)0.20147304 (±1.93402481)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-14-28-42
Dimensions454545
Spacing454545
CellA=B=C: 126.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z454545
origin x/y/z0.0000.0000.000
length x/y/z126.000126.000126.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128168
MAP C/R/S123
start NC/NR/NS-28-14-42
NC/NR/NS454545
D min/max/mean-7.3376.9420.201

-
Supplemental data

-
Sample components

-
Entire : 13-protofilament microtubule-bound human kinesin-5 motor domain w...

EntireName: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP bound. A gold cluster is attached to the neck linker (V365C).
Components
  • Sample: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP bound. A gold cluster is attached to the neck linker (V365C).
  • Protein or peptide: alpha tubulin
  • Protein or peptide: beta tubulin
  • Protein or peptide: Kinesin-5 motor domain

-
Supramolecule #1000: 13-protofilament microtubule-bound human kinesin-5 motor domain w...

SupramoleculeName: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP bound. A gold cluster is attached to the neck linker (V365C).
type: sample / ID: 1000
Oligomeric state: 13-protofilament microtubule with one kinesin-5 motor domain bound every tubulin heterodimers
Number unique components: 3

-
Macromolecule #1: alpha tubulin

MacromoleculeName: alpha tubulin / type: protein_or_peptide / ID: 1 / Name.synonym: TUBULIN ALPHA-1D CHAIN / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: Cattle / Tissue: brain
SequenceInterPro: Alpha tubulin

-
Macromolecule #2: beta tubulin

MacromoleculeName: beta tubulin / type: protein_or_peptide / ID: 2 / Name.synonym: TUBULIN BETA-2B CHAIN / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: Cattle / Tissue: brain
SequenceInterPro: Beta tubulin, autoregulation binding site

-
Macromolecule #3: Kinesin-5 motor domain

MacromoleculeName: Kinesin-5 motor domain / type: protein_or_peptide / ID: 3 / Name.synonym: KINESIN-LIKE PROTEIN KIF11
Details: undecagold cluster was attached to the specific cysteine residue in the neck linker (V365C)
Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET21a
SequenceInterPro: Kinesin motor domain, conserved site

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

BufferpH: 6.8 / Details: 80 mM PIPES, 5 mM MgCl2, 1 mM EGTA, 5 mM AMPPNP
GridDetails: 400 mesh holey carbon grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I / Method: chamber at 24 degrees C, blot 2.5 sec

-
Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 90 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
DateNov 26, 2011
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 35 / Average electron dose: 18 e/Å2 / Bits/pixel: 8
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

-
Image processing

CTF correctionDetails: FREALIGN
Final reconstructionResolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, FREALIGN
Details: Approximately 21,000 asymmetric units were averaged in the final reconstruction. The deposited map is low pass filtered at 16 A.
Number images used: 1753
DetailsThe particles were selected along individual microtubules.

+
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