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- PDB-7rs5: Cryo-EM structure of Kip3 (AMPPNP) bound to Taxol-Stabilized Micr... -

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Basic information

Entry
Database: PDB / ID: 7rs5
TitleCryo-EM structure of Kip3 (AMPPNP) bound to Taxol-Stabilized Microtubules
Components
  • Tubulin alpha-1A chain
  • Tubulin beta chain
  • yeast kinesin-8/ Kip3
KeywordsMOTOR PROTEIN / kinesin-8 / microtubules / complex
Function / homology
Function and homology information


structural constituent of cytoskeleton / microtubule cytoskeleton organization / mitotic cell cycle / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
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 ...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
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / Tubulin alpha-1A chain / Tubulin beta chain
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsHernandez-Lopez, R.A. / Leschziner, A.E. / Arellano-Santoyo, H. / Pellman, D. / Stokasimov, E. / Wang, R.Y.-R.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Biorxiv / Year: 2021
Title: Multimodal tubulin binding by the yeast kinesin-8, Kip3, underlies its motility and depolymerization
Authors: Arellano-Santoyo, H. / Hernandez-Lopez, R.A. / Stokasimov, E. / Wang, R.Y.R. / Pellman, D. / Leschziner, A.E.
History
DepositionAug 10, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 17, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 8, 2023Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year / _citation_author.name
Revision 1.2Jun 5, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tubulin alpha-1A chain
B: Tubulin beta chain
K: yeast kinesin-8/ Kip3
C: Tubulin alpha-1A chain
D: Tubulin beta chain
a: yeast kinesin-8/ Kip3
E: Tubulin alpha-1A chain
F: Tubulin beta chain
b: yeast kinesin-8/ Kip3
G: Tubulin alpha-1A chain
H: Tubulin beta chain
c: yeast kinesin-8/ Kip3
I: Tubulin alpha-1A chain
J: Tubulin beta chain
d: yeast kinesin-8/ Kip3
L: Tubulin alpha-1A chain
M: Tubulin beta chain
e: yeast kinesin-8/ Kip3
N: Tubulin alpha-1A chain
O: Tubulin beta chain
f: yeast kinesin-8/ Kip3
P: Tubulin alpha-1A chain
Q: Tubulin beta chain
g: yeast kinesin-8/ Kip3
R: Tubulin alpha-1A chain
S: Tubulin beta chain
h: yeast kinesin-8/ Kip3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,280,61481
Polymers1,259,23827
Non-polymers21,37654
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 27 molecules ACEGILNPRBDFHJMOQSKabcdefgh

#1: Protein
Tubulin alpha-1A chain / Alpha-tubulin 1 / Tubulin alpha-1 chain


Mass: 50107.238 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02550
#2: Protein
Tubulin beta chain / Beta-tubulin


Mass: 49907.770 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02554
#3: Protein
yeast kinesin-8/ Kip3


Mass: 39900.332 Da / Num. of mol.: 9
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Saccharomyces cerevisiae (brewer's yeast)

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Non-polymers , 5 types, 54 molecules

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


Mass: 523.180 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, energy-carrying molecule*YM
#7: Chemical
ChemComp-TA1 / TAXOL


Mass: 853.906 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C47H51NO14 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication, chemotherapy*YM
#8: Chemical
ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM

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Details

Has ligand of interestY

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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

Component
IDNameTypeEntity IDParent-IDSource
1Cryo-EM structure of Kip3 (AMPPNP) bound to Taxol-Stabilized MicrotubulesCOMPLEX#1-#30MULTIPLE SOURCES
2Taxol-stabilized microtubulesCOMPLEX#1-#21NATURAL
3yeast kinesin-8/ Kip3COMPLEX#31RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Sus scrofa (pig)9823
23Saccharomyces cerevisiae (brewer's yeast)4932
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 8
Details: cryoEM buffer (50 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT supplemented with 2mM AMPPNP)
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Highly 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. To ...Details: Highly 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. To prepare Taxol-stabilized microtubules, tubulin was polymerized with a stepwise addition of Taxol as follows: 20 uL of tubulin stock was thawed quickly and placed on ice. 10 uL of BRB80 supplemented with 3 mM GTP were added and the mixture was transferred to a 37 C water bath. After 15, 30, and 45 minutes, additions of 0.5, 0.5, and 1.0 uL of 2 mM Taxol were added by gentle swirling. The mixture was then incubated for an additional 1 h at 37C. 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 2 mM 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.
Specimen supportGrid material: COPPER / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 22 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Images were recorded using a semi-automated acquisition program Serial EM with a defocus range from 1.5 to 3.5 um.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1194
Details: Final accumulated electron doses were 40 electrons/A2. Images were collected in super-resolution mode. The total exposure time was 4 seconds, fractionated into 20 subframes, each with an exposure time of 0.2 s.

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Processing

EM software
IDNameVersionCategory
1Appionparticle selection
4CTFFIND4CTF correction
7Rosettamodel fitting
9Rosettamodel refinement
10EMAN2initial Euler assignment
11FREALIGNfinal Euler assignment
13FREALIGN3D reconstruction
CTF correctionDetails: The contrast transfer function (CTF) was estimated using CTFFIND4 [(Rohou and Grigorieff, 2015) CTFFIND4: Fast and accurate] with a 500 um step search. A second CTFFIND run with a 100 um ...Details: The contrast transfer function (CTF) was estimated using CTFFIND4 [(Rohou and Grigorieff, 2015) CTFFIND4: Fast and accurate] with a 500 um step search. A second CTFFIND run with a 100 um step search was carried out to refine the initial defocus values. Micrographs whose estimated resolution was lower than 8 Angstrom with 0.8 confidence were excluded.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -25.76 ° / Axial rise/subunit: 8.55 Å / Axial symmetry: C14
Particle selectionNum. of particles selected: 67040
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.
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 14934
Details: Pseudo-helical symmetry was applied during the reconstruction step
Symmetry type: HELICAL
Atomic model buildingB value: 100 / Protocol: OTHER / Space: REAL
Target criteria: Overall correlation of the residues to the map
Details: Multiple 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 ...Details: Multiple 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.
Atomic model building

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

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
13JAT

3jat

A3JAT1
23JAT

3jat

B3JAT1
34FRZ

4frz

K4FRZ2

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