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- PDB-6e8g: CryoEM reconstruction of IST1-CHMP1B copolymer filament bound to ... -

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Basic information

Entry
Database: PDB / ID: 6e8g
TitleCryoEM reconstruction of IST1-CHMP1B copolymer filament bound to ssDNA at 2.9 Angstrom resolution
Components
  • Charged multivesicular body protein 1b
  • IST1 homolog
KeywordsDNA BINDING PROTEIN / PROTEIN FIBRIL / ESCRT-III / CHMP1B / IST1 / ssDNA
Function / homology
Function and homology information


MIT domain binding / multivesicular body-lysosome fusion / amphisome membrane / vesicle fusion with vacuole / ESCRT III complex disassembly / late endosome to lysosome transport / ESCRT III complex / kinetochore microtubule / endosome transport via multivesicular body sorting pathway / cytoskeleton-dependent cytokinesis ...MIT domain binding / multivesicular body-lysosome fusion / amphisome membrane / vesicle fusion with vacuole / ESCRT III complex disassembly / late endosome to lysosome transport / ESCRT III complex / kinetochore microtubule / endosome transport via multivesicular body sorting pathway / cytoskeleton-dependent cytokinesis / collateral sprouting / membrane coat / regulation of centrosome duplication / nuclear membrane reassembly / Sealing of the nuclear envelope (NE) by ESCRT-III / multivesicular body sorting pathway / positive regulation of collateral sprouting / midbody abscission / membrane fission / plasma membrane repair / late endosome to vacuole transport / ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / multivesicular body assembly / multivesicular body membrane / regulation of mitotic spindle assembly / Flemming body / mitotic metaphase chromosome alignment / nucleus organization / viral budding via host ESCRT complex / endoplasmic reticulum-Golgi intermediate compartment / autophagosome membrane / positive regulation of proteolysis / autophagosome maturation / nuclear pore / multivesicular body / viral budding from plasma membrane / establishment of protein localization / kinetochore / autophagy / azurophil granule lumen / intracellular protein localization / nuclear envelope / protein transport / midbody / endosome membrane / cadherin binding / protein domain specific binding / lysosomal membrane / cell division / intracellular membrane-bounded organelle / centrosome / Neutrophil degranulation / chromatin / protein-containing complex binding / extracellular exosome / extracellular region / nucleoplasm / identical protein binding / plasma membrane / cytosol
Similarity search - Function
Vacuolar protein sorting-associated protein Ist1 / Vacuolar protein sorting-associated protein IST1-like / Regulator of Vps4 activity in the MVB pathway / Snf7 family / Snf7
Similarity search - Domain/homology
IST1 homolog / Charged multivesicular body protein 1b
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsTalledge, N. / Frost, A. / McCullough, J.
Funding support United States, 8items
OrganizationGrant numberCountry
National Institutes of Health/Office of the DirectorS10OD020054 United States
National Institutes of Health/Office of the Director1S10OD021741 United States
National Institutes of Health/Office of the Director1S10OD021596-01 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1DP2GM110772-01 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)3P50GM082545-07S1 United States
Howard Hughes Medical Institute (HHMI) United States
American Asthma Foundation United States
Chan Zuckerberg Biohub United States
CitationJournal: Biorxiv / Year: 2018
Title: The ESCRT-III proteins IST1 and CHMP1B assemble around nucleic acids
Authors: Talledge, N. / McCullough, J. / Wenzel, D.M. / Nguyen, H.C. / Lalonde, M. / Bajorek, M. / Skalicky, J.J. / Frost, A. / Sundquist, W.I.
History
DepositionJul 29, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 15, 2018Provider: repository / Type: Initial release
Revision 1.1Feb 20, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 13, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2
Item: _citation.journal_id_ISSN / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
S: IST1 homolog
T: Charged multivesicular body protein 1b
A: IST1 homolog
B: Charged multivesicular body protein 1b
C: IST1 homolog
D: Charged multivesicular body protein 1b
E: IST1 homolog
F: Charged multivesicular body protein 1b
G: IST1 homolog
H: Charged multivesicular body protein 1b
I: IST1 homolog
J: Charged multivesicular body protein 1b
K: IST1 homolog
L: Charged multivesicular body protein 1b
M: IST1 homolog
N: Charged multivesicular body protein 1b
O: IST1 homolog
P: Charged multivesicular body protein 1b
Q: IST1 homolog
R: Charged multivesicular body protein 1b
V: IST1 homolog
W: Charged multivesicular body protein 1b
X: IST1 homolog
Y: Charged multivesicular body protein 1b
Z: IST1 homolog
AA: Charged multivesicular body protein 1b
BA: IST1 homolog
CA: Charged multivesicular body protein 1b
DA: IST1 homolog
EA: Charged multivesicular body protein 1b
FA: IST1 homolog
GA: Charged multivesicular body protein 1b
HA: IST1 homolog
IA: Charged multivesicular body protein 1b
JA: IST1 homolog
KA: Charged multivesicular body protein 1b
LA: IST1 homolog
MA: Charged multivesicular body protein 1b
NA: IST1 homolog
OA: Charged multivesicular body protein 1b
PA: IST1 homolog
QA: Charged multivesicular body protein 1b
RA: IST1 homolog
SA: Charged multivesicular body protein 1b
TA: IST1 homolog
UA: Charged multivesicular body protein 1b
VA: IST1 homolog
WA: Charged multivesicular body protein 1b
XA: IST1 homolog
YA: Charged multivesicular body protein 1b
ZA: IST1 homolog
AB: Charged multivesicular body protein 1b
BB: IST1 homolog
CB: Charged multivesicular body protein 1b
DB: IST1 homolog
EB: Charged multivesicular body protein 1b
FB: IST1 homolog
GB: Charged multivesicular body protein 1b
HB: IST1 homolog
IB: Charged multivesicular body protein 1b
JB: IST1 homolog
KB: Charged multivesicular body protein 1b
LB: IST1 homolog
MB: Charged multivesicular body protein 1b
NB: IST1 homolog
OB: Charged multivesicular body protein 1b
PB: IST1 homolog
QB: Charged multivesicular body protein 1b
RB: IST1 homolog
SB: Charged multivesicular body protein 1b
TB: IST1 homolog
UB: Charged multivesicular body protein 1b


Theoretical massNumber of molelcules
Total (without water)2,237,94272
Polymers2,237,94272
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, Purified recombinant protein was verified by mass spectrometry verification.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area347580 Å2
ΔGint-2205 kcal/mol
Surface area543190 Å2
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 36 / Rise per n subunits: 3.17 Å / Rotation per n subunits: 21.16 °)

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Components

#1: Protein ...
IST1 homolog / hIST1 / Putative MAPK-activating protein PM28


Mass: 40024.711 Da / Num. of mol.: 36
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IST1, KIAA0174 / Plasmid: pET16b / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) RIPL / References: UniProt: P53990
#2: Protein ...
Charged multivesicular body protein 1b / CHMP1.5 / Chromatin-modifying protein 1b / CHMP1b / Vacuolar protein sorting-associated protein 46- ...CHMP1.5 / Chromatin-modifying protein 1b / CHMP1b / Vacuolar protein sorting-associated protein 46-2 / hVps46-2


Mass: 22140.354 Da / Num. of mol.: 36 / Mutation: K37E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CHMP1B, C18orf2 / Plasmid: pGEX / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) RIPL / References: UniProt: Q7LBR1

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Helical assembly of IST1 and CHMP1B protein bound to ssDNA
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Homo sapiens (human) / Cellular location: cytoplasm
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) RIPL
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1125 mMsodium chlorideNaCl1
225 mMTrisC4H11NO31
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 292.15 K / Details: 0 mm offset with 10 sec wait time and 2-4 sec blot

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 29000 X / Cs: 2.6 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6.2 sec. / Electron dose: 62 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: 2980
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 32 / Used frames/image: 3-32

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Processing

EM software
IDNameVersionCategoryDetails
1RELION2.1particle selectionManual helix picking was used to determine the filaments helical path.
3SerialEMimage acquisition
5Gctf1.06CTF correctionHigh resolution refinement between 15 and 4 Angstroms was performed.
8UCSF Chimeramodel fitting
9Cootmodel fitting
13RELION2.1initial Euler assignmentrelion_refine
15RELION2.1final Euler assignment
17RELION2.1classification
19RELION2.13D reconstruction
21PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 21.16 ° / Axial rise/subunit: 3.17 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 224252
Details: Helical particle segments were selected using RELION manualpick and extracted using overlapping segments with an asymmetric unit of 17 subunits with a 3.0 Angstrom rise (51 Angstrom total).
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 101990 / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingB value: 52.5 / Protocol: AB INITIO MODEL / Space: REAL

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