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- PDB-8wzm: Human erythrocyte catalase with CTAB as additive during EM sample... -

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Basic information

Entry
Database: PDB / ID: 8wzm
TitleHuman erythrocyte catalase with CTAB as additive during EM sample 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 L-ascorbic acid / response to ozone / 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 L-ascorbic acid / response to ozone / 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 / peroxisome / response to estradiol / 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: 2.7 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 2, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 10, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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

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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
20.002 %CTAB1
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 R1.2/1.3
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: 130000 X / Calibrated magnification: 47619 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: 8 sec. / Electron dose: 40.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter name: GIF Bioquantum / Details: Images were collected in EFTEM mode. / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 32

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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: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 153336 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 81.25 / Protocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 1dgf

1dgf


Accession code: 1dgf / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00616892
ELECTRON MICROSCOPYf_angle_d0.63623048
ELECTRON MICROSCOPYf_dihedral_angle_d4.7489940
ELECTRON MICROSCOPYf_chiral_restr0.052340
ELECTRON MICROSCOPYf_plane_restr0.0053056

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