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- PDB-8wzh: Human erythrocyte catalase with SLS as additive during cryo-EM gr... -

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Basic information

Entry
Database: PDB / ID: 8wzh
TitleHuman erythrocyte catalase with SLS as additive during cryo-EM grid preparation
ComponentsCatalase
KeywordsOXIDOREDUCTASE / Catalase / heme / NADP
Function / homology
Function and homology information


response to phenylpropanoid / aminoacylase activity / catalase complex / hemoglobin metabolic process / response to inactivity / cellular detoxification of hydrogen peroxide / response to ozone / response to L-ascorbic acid / oxidoreductase activity, acting on peroxide as acceptor / response to light intensity ...response to phenylpropanoid / aminoacylase activity / catalase complex / hemoglobin metabolic process / response to inactivity / cellular detoxification of hydrogen peroxide / response to ozone / response to L-ascorbic acid / oxidoreductase activity, acting on peroxide as acceptor / response to light intensity / catalase / UV protection / response to fatty acid / response to vitamin A / catalase activity / peroxisomal membrane / ureteric bud development / triglyceride metabolic process / Detoxification of Reactive Oxygen Species / antioxidant activity / peroxisomal matrix / positive regulation of cell division / response to vitamin E / response to hyperoxia / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / response to cadmium ion / aerobic respiration / cholesterol metabolic process / response to activity / response to reactive oxygen species / hydrogen peroxide catabolic process / Peroxisomal protein import / response to lead ion / response to insulin / response to hydrogen peroxide / cellular response to growth factor stimulus / osteoblast differentiation / response to estradiol / peroxisome / NADP binding / secretory granule lumen / response to ethanol / ficolin-1-rich granule lumen / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / response to hypoxia / response to xenobiotic stimulus / intracellular membrane-bounded organelle / focal adhesion / heme binding / Neutrophil degranulation / negative regulation of apoptotic process / enzyme binding / protein homodimerization activity / protein-containing complex / mitochondrion / extracellular exosome / extracellular region / identical protein binding / membrane / metal ion binding / cytoplasm / cytosol
Similarity search - Function
Catalase, clade 3 / Catalase, mono-functional, haem-containing, clades 1 and 3 / Catalase haem-binding site / Catalase proximal heme-ligand signature. / Catalase / Catalase active site / Catalase proximal active site signature. / Catalase immune-responsive domain / Catalase-related immune-responsive / Catalase core domain ...Catalase, clade 3 / Catalase, mono-functional, haem-containing, clades 1 and 3 / Catalase haem-binding site / Catalase proximal heme-ligand signature. / Catalase / Catalase active site / Catalase proximal active site signature. / Catalase immune-responsive domain / Catalase-related immune-responsive / Catalase core domain / Catalase, mono-functional, haem-containing / Catalase / catalase family profile. / Catalase superfamily
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / Chem-NDP / Catalase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsYadav, S. / Vinothkumar, K.R.
Funding support India, 2items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014 India
Other governmentRTI4006 India
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2024
Title: Factors affecting macromolecule orientations in thin films formed in cryo-EM.
Authors: Swati Yadav / Kutti R Vinothkumar /
Abstract: The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the ...The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge-freeze method first described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air-water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, the effect of physical and chemical factors on the orientations of macromolecules was investigated through an analysis of selected well studied macromolecules, and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of the interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and to provide a framework to design small-molecule additives to enhance sample stability and behaviour.
History
DepositionNov 1, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 10, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Catalase
A: Catalase
C: Catalase
D: Catalase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)244,79612
Polymers239,3484
Non-polymers5,4488
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11B
21A
12B
22C
13B
23D
14A
24C
15A
25D
16C
26D

NCS domain segments:

Component-ID: _ / Beg auth comp-ID: ARG / Beg label comp-ID: ARG / End auth comp-ID: ASN / End label comp-ID: ASN / Refine code: _ / Auth seq-ID: 5 - 501 / Label seq-ID: 5 - 501

Dom-IDEns-IDAuth asym-IDLabel asym-ID
11BA
21AB
12BA
22CC
13BA
23DD
14AB
24CC
15AB
25DD
16CC
26DD

NCS ensembles :
ID
1
2
3
4
5
6

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Components

#1: Protein
Catalase


Mass: 59836.996 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P04040, catalase
#2: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE


Mass: 745.421 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C21H30N7O17P3 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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

ComponentName: Catalase purified from human erythrocytes / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.238 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
150 mMTris1
20.04 %SLS1
SpecimenConc.: 3.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: Blot force, 0

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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: 75000 X / Calibrated magnification: 130841 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 33 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1
Details: Images were collected in movie mode at 40 frames per second. Total of 25 frames were saved for each movie.
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
5RELION3.1CTF correction
8UCSF Chimera1.15model fitting
9Coot0.9.5model fitting
14RELION3.13D reconstruction
21PHENIX1.15model refinement
22REFMAC5.8.0411model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33241 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 151.4 / Protocol: OTHER / Space: RECIPROCAL
Atomic model buildingPDB-ID: 1dgf

1dgf


Accession code: 1dgf / Source name: PDB / Type: experimental model
RefinementResolution: 3.7→121.98 Å / Cor.coef. Fo:Fc: 0.934 / SU B: 51.884 / SU ML: 0.759
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.36617 --
obs0.36617 53627 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 138.859 Å2
Refinement stepCycle: 1 / Total: 16372
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0040.01316893
ELECTRON MICROSCOPYr_bond_other_d0.030.01714737
ELECTRON MICROSCOPYr_angle_refined_deg1.1791.67423048
ELECTRON MICROSCOPYr_angle_other_deg1.2461.58834269
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.57151984
ELECTRON MICROSCOPYr_dihedral_angle_2_deg35.25522.339992
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.36152584
ELECTRON MICROSCOPYr_dihedral_angle_4_deg18.03115112
ELECTRON MICROSCOPYr_chiral_restr0.0510.22080
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.0219228
ELECTRON MICROSCOPYr_gen_planes_other0.0020.023760
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it0.65815.127948
ELECTRON MICROSCOPYr_mcbond_other0.65815.1197947
ELECTRON MICROSCOPYr_mcangle_it1.24822.6789928
ELECTRON MICROSCOPYr_mcangle_other1.24822.6799929
ELECTRON MICROSCOPYr_scbond_it0.31315.0748945
ELECTRON MICROSCOPYr_scbond_other0.31315.0748943
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other0.67722.58113121
ELECTRON MICROSCOPYr_long_range_B_refined5.23968471
ELECTRON MICROSCOPYr_long_range_B_other5.23968472
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Rms dev position: 0 Å / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumber
11B33100
12A33100
21B33100
22C33100
31B33102
32D33102
41A33098
42C33098
51A33100
52D33100
61C33102
62D33102
LS refinement shellResolution: 3.7→3.796 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.803 3967 -
obs--100 %

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