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- PDB-7xoj: Cryo-EM structure of GroEL bound to unfolded substrate (UGT1A) at... -

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Basic information

Entry
Database: PDB / ID: 7xoj
TitleCryo-EM structure of GroEL bound to unfolded substrate (UGT1A) at 2.8 Ang. resolution (Consensus Refinement)
ComponentsChaperonin GroEL
KeywordsCHAPERONE / cryogenic electron microscopy / single-particle analysis / molecular motion / structure-function relationship / focus classification / separating heterogeneity / groel / unfolded protein
Function / homology
Function and homology information


GroEL-GroES complex / chaperonin ATPase / virion assembly / : / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / response to heat ...GroEL-GroES complex / chaperonin ATPase / virion assembly / : / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / response to heat / protein refolding / magnesium ion binding / ATP hydrolysis activity / ATP binding / identical protein binding / membrane / cytosol
Similarity search - Function
GroEL / GroEL / Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like apical domain superfamily / TCP-1/cpn60 chaperonin family / Chaperonin Cpn60/GroEL/TCP-1 family ...GroEL / GroEL / Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like apical domain superfamily / TCP-1/cpn60 chaperonin family / Chaperonin Cpn60/GroEL/TCP-1 family / 3-Layer(bba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsStapleton, K. / Takagi, J. / Mizohata, E.
Funding support Japan, 7items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)21am0101075 Japan
Japan Agency for Medical Research and Development (AMED)22ama121025j0001 Japan
Japan Agency for Medical Research and Development (AMED)JP21am0101066 Japan
Japan Society for the Promotion of Science (JSPS)JP19K06513 Japan
Japan Science and TechnologyJPMJPR17GB Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)19H05780 Japan
New Energy and Industrial Technology Development Organization (NEDO)JPNP18016 Japan
CitationJournal: To Be Published
Title: Unmasking GroEL: Structure, dynamics, and substrate binding revealed by single-particle cryo-EM
Authors: Stapleton, K.M. / Mizobata, T. / Miyazaki, N. / Takatsuji, T. / Kato, T. / Iwasaki, K. / Standley, D.M. / Kawamura, T. / Nakane, T. / Takagi, J. / Mizohata, E.
History
DepositionMay 1, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 3, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Chaperonin GroEL
B: Chaperonin GroEL
C: Chaperonin GroEL
D: Chaperonin GroEL
E: Chaperonin GroEL
F: Chaperonin GroEL
G: Chaperonin GroEL
H: Chaperonin GroEL
I: Chaperonin GroEL
J: Chaperonin GroEL
K: Chaperonin GroEL
L: Chaperonin GroEL
M: Chaperonin GroEL
N: Chaperonin GroEL


Theoretical massNumber of molelcules
Total (without water)801,64714
Polymers801,64714
Non-polymers00
Water00
1


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

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Components

#1: Protein
Chaperonin GroEL / 60 kDa chaperonin / Chaperonin-60 / Cpn60 / GroEL protein


Mass: 57260.504 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: groEL, groL, mopA, b4143, JW4103 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A6F5, chaperonin ATPase

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Experimental details

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Experiment

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

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1GroEL-UGT1A complexCOMPLEXGroEL Complexed with Unfolded UGT1Aall0RECOMBINANT
2GroELCOMPLEXThe model was built in a map at 2.8 A global resolution by FSC 0.143all1RECOMBINANT
3UDP-glucuronosyltransferase 1A (UGT1A)COMPLEXModel was not built because of weak density map1RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
21Escherichia coli (E. coli)562pKY206
32Escherichia coli (E. coli)562pKY206
43Escherichia coli (E. coli)562pTAT6
Buffer solutionpH: 7.5
Details: Sample containing GorEL-UGT1A was made fresh and used without undergoing any freeze-thaw cycles to avoid degradation in the solution. The sample was in a buffer solution of 150mM NaCl, 20mM Tris-HCl at pH 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 nMsodium chlorideNaCl1
220 mMTris Hydrochloride AcidTris-HCl1
SpecimenConc.: 33 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: 3 ul of 33 mg/ml GroEL-UGT1A was placed on Holey carbon Quanitifoil copper grids (300 mesh size R1.2/1.3) and blotted for 3 seconds (blot force =1)

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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 defocus max: 2000 nm / Nominal defocus min: 1200 nm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2951
Details: Using a Titan KRIOS TEM operated at 300 kV, 2,951 movies were collected using the Falcon III DED in Counting mode at a magnification of 75000x corresponding to a pixel size of 0.87 Pixel/A ...Details: Using a Titan KRIOS TEM operated at 300 kV, 2,951 movies were collected using the Falcon III DED in Counting mode at a magnification of 75000x corresponding to a pixel size of 0.87 Pixel/A at the specimen level. All 2,951 movies were imported into the RELION pipeline and prepared for single-particle analysis
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION3.1particle selectionLaplacian Of Gaussian (LoG)
2EPUimage acquisitionUsed to callobrate and collect raw movies
3RELION3.1image acquisitionAll processing of movies and particles was performed in RELION
5CTFFIND4.1CTF correction
8UCSF ChimeraX1.2.5model fitting
10RELION3.1initial Euler assignment
13RELION3.13D reconstruction
14PHENIX1.19.2-4158model refinement
CTF correctionDetails: Using RELIONs own implementation of CTFFIND4.1 --Box 512 --ResMin 30 --ResMax 2 --dFMin 5000 --dFMax 50000 --FStep 250 --dAst 100
Type: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 1289236
Details: Parameters for auto picking (LoG) single particles from motion-corrected micrographs with estimated CTF parameters resulted in 1,289,236 particles picked (average of 437 particles per ...Details: Parameters for auto picking (LoG) single particles from motion-corrected micrographs with estimated CTF parameters resulted in 1,289,236 particles picked (average of 437 particles per micrograph). All particle images were then extracted with a box size of 300 pixels and a spherical mask diameter of 240 A for 2D classification.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1202095 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 91 / Protocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00354362
ELECTRON MICROSCOPYf_angle_d0.46173402
ELECTRON MICROSCOPYf_dihedral_angle_d3.5527728
ELECTRON MICROSCOPYf_chiral_restr0.0398932
ELECTRON MICROSCOPYf_plane_restr0.0039618

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