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- EMDB-33354: Cryo-EM structure of empty ring subunit 2 (ER2) from GroEL-UGT1A ... -

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Basic information

Entry
Database: EMDB / ID: EMD-33354
TitleCryo-EM structure of empty ring subunit 2 (ER2) from GroEL-UGT1A single empty ring complex
Map dataPrimary EM-map of GroEL-UGT1A for Subunit ER2 (unmasked)
Sample
  • Complex: GroEL-UGT1A single empty ring complex
    • Complex: Empty Ring subunit 2 (ER-2) of GroEL
      • Protein or peptide: Chaperonin GroEL
    • Complex: UDP-glucuronosyltransferase 1A (UGT1A)
Function / homology
Function and homology information


GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / 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 / chaperone cofactor-dependent protein refolding / 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 / membrane / identical protein binding / cytosol
Similarity search - Function
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
Similarity search - Domain/homology
Biological speciesEscherichia coli (E. coli) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsStapleton K / Takagi J / Mizohata E
Funding support Japan, 7 items
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 KM / Mizobata T / Miyazaki N / Takatsuji T / Kato T / Iwasaki K / Standley DM / Kawamura T / Nakane T / Takagi J / Mizohata E
History
DepositionMay 1, 2022-
Header (metadata) releaseMay 3, 2023-
Map releaseMay 3, 2023-
UpdateMay 3, 2023-
Current statusMay 3, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33354.png

Downloads & links

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Map

FileDownload / File: emd_33354.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary EM-map of GroEL-UGT1A for Subunit ER2 (unmasked)
Voxel sizeX=Y=Z: 0.87 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.030190399 - 0.09386367
Average (Standard dev.)-0.00015989969 (±0.0057826894)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 261.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_33354_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_33354_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Additional EM-map of a GroEL subunit that was...

Fileemd_33354_additional_1.map
AnnotationAdditional EM-map of a GroEL subunit that was used for modeling the atomic coordinates (subunit ER2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map-1 used for processing the Primary EM-map of...

Fileemd_33354_half_map_1.map
AnnotationHalf_map-1 used for processing the Primary EM-map of GroEL-UGT1A for Subunit ER2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map-2 used for processing the Primary EM-map of...

Fileemd_33354_half_map_2.map
AnnotationHalf_map-2 used for processing the Primary EM-map of GroEL-UGT1A for Subunit ER2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : GroEL-UGT1A single empty ring complex

EntireName: GroEL-UGT1A single empty ring complex
Components
  • Complex: GroEL-UGT1A single empty ring complex
    • Complex: Empty Ring subunit 2 (ER-2) of GroEL
      • Protein or peptide: Chaperonin GroEL
    • Complex: UDP-glucuronosyltransferase 1A (UGT1A)

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Supramolecule #1: GroEL-UGT1A single empty ring complex

SupramoleculeName: GroEL-UGT1A single empty ring complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #2: Empty Ring subunit 2 (ER-2) of GroEL

SupramoleculeName: Empty Ring subunit 2 (ER-2) of GroEL / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: all
Details: The model was built in the additional EM-map of ER2 (masked)
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: UDP-glucuronosyltransferase 1A (UGT1A)

SupramoleculeName: UDP-glucuronosyltransferase 1A (UGT1A) / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Details: Model is not built
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Chaperonin GroEL

MacromoleculeName: Chaperonin GroEL / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: chaperonin ATPase
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 57.260504 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: AAKDVKFGND ARVKMLRGVN VLADAVKVTL GPKGRNVVLD KSFGAPTITK DGVSVAREIE LEDKFENMGA QMVKEVASKA NDAAGDGTT TATVLAQAII TEGLKAVAAG MNPMDLKRGI DKAVTAAVEE LKALSVPCSD SKAIAQVGTI SANSDETVGK L IAEAMDKV ...String:
AAKDVKFGND ARVKMLRGVN VLADAVKVTL GPKGRNVVLD KSFGAPTITK DGVSVAREIE LEDKFENMGA QMVKEVASKA NDAAGDGTT TATVLAQAII TEGLKAVAAG MNPMDLKRGI DKAVTAAVEE LKALSVPCSD SKAIAQVGTI SANSDETVGK L IAEAMDKV GKEGVITVED GTGLQDELDV VEGMQFDRGY LSPYFINKPE TGAVELESPF ILLADKKISN IREMLPVLEA VA KAGKPLL IIAEDVEGEA LATLVVNTMR GIVKVAAVKA PGFGDRRKAM LQDIATLTGG TVISEEIGME LEKATLEDLG QAK RVVINK DTTTIIDGVG EEAAIQGRVA QIRQQIEEAT SDYDREKLQE RVAKLAGGVA VIKVGAATEV EMKEKKARVE DALH ATRAA VEEGVVAGGG VALIRVASKL ADLRGQNEDQ NVGIKVALRA MEAPLRQIVL NCGEEPSVVA NTVKGGDGNY GYNAA TEEY GNMIDMGILD PTKVTRSALQ YAASVAGLMI TTECMVTDLP KNDAADLGAA GGMGGMGGMG GMM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration33 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chlorideSodium Cloride
20.0 mMTris-HClTrisTris Hydrochloride Acid

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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.2 µm
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2951 / Average electron dose: 40.0 e/Å2
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber 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 (an 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 (an 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.
Startup modelType of model: INSILICO MODEL
Details: The initial model was from a previously derived map generated from screening data of the same sample and grid
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Details: D7 symmetry expanded particle set / Number images used: 144509
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7xoo:
Cryo-EM structure of empty ring subunit 2 (ER2) from GroEL-UGT1A single empty ring complex

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