|Entry||Database: PDB / ID: 6nbc|
|Title||human methemoglobin state 1 determined using single-particle cryo-EM at 200 keV|
|Keywords||OXYGEN TRANSPORT / heme-binding / hetero-4-mer / globin|
|Function / homology|
Function and homology information
nitric oxide transport / hemoglobin binding / hemoglobin alpha binding / haptoglobin-hemoglobin complex / organic acid binding / renal absorption / cellular oxidant detoxification / oxygen transport / hemoglobin complex / endocytic vesicle lumen ...nitric oxide transport / hemoglobin binding / hemoglobin alpha binding / haptoglobin-hemoglobin complex / organic acid binding / renal absorption / cellular oxidant detoxification / oxygen transport / hemoglobin complex / endocytic vesicle lumen / positive regulation of cell death / platelet aggregation / hydrogen peroxide catabolic process / regulation of blood pressure / cytosolic small ribosomal subunit / response to hydrogen peroxide / oxygen carrier activity / bicarbonate transport / oxygen binding / receptor-mediated endocytosis / positive regulation of nitric oxide biosynthetic process / tertiary granule lumen / ficolin-1-rich granule lumen / blood microparticle / blood coagulation / iron ion binding / neutrophil degranulation / heme binding / extracellular space / extracellular exosome / membrane / extracellular region / metal ion binding / cytosol
Haemoglobin, beta-type / Haemoglobin, alpha-type / Globin / Globin/Protoglobin / Globin / Globin-like superfamily / Haemoglobin, pi / Globins / Globin-like / Orthogonal Bundle / Mainly Alpha
Hemoglobin subunit beta / Hemoglobin subunit alpha
|Biological species||Homo sapiens (human)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å|
|Authors||Herzik Jr., M.A. / Wu, M. / Lander, G.C.|
|Funding support|| United States, 1items |
|Citation||Journal: Nat Commun / Year: 2019|
Title: High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM.
Authors: Mark A Herzik / Mengyu Wu / Gabriel C Lander /
Abstract: Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While ...Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While the Volta phase plate has enabled visualization of specimens in this size range, this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.7 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity to enable investigation of drug-target interactions. Our ~2.8 Å and ~3.2 Å structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the sub-nanometer cryo-EM structure of a sub-50 kDa protein.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
Downloads & links
A: Hemoglobin subunit alpha
B: Hemoglobin subunit beta
C: Hemoglobin subunit alpha
D: Hemoglobin subunit beta
|Number of models||4|
Mass: 14993.159 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
Plasmid details: Obtained in lyophilized form from Sigma Aldrich
References: UniProt: P69905
Mass: 15459.697 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
Plasmid details: Obtained in lyophilized form from Sigma Aldrich
References: UniProt: P68871
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: methemoglobin from human / Type: COMPLEX|
Details: Lyophilized human methemoglobin purchased from Sigma Aldrich
Entity ID: 1, 2 / Source: NATURAL
|Molecular weight||Value: 0.064 MDa / Experimental value: NO|
|Source (natural)||Organism: Homo sapiens (human)|
|Buffer solution||pH: 7.5|
|Specimen||Conc.: 12 mg/ml|
Details: Lyophilized human methemoglobin (Sigma Aldrich) was solubilized.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
|Specimen support||Details: Grids were plasma cleaned using a Solarus plasma cleaner (Gatan, Inc.).|
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 K|
Details: Sample was manually blotted for 4-5 seconds using Whatman No. 1 filter paper immediately prior to plunge-freezing.
-Electron microscopy imaging
Model: Talos Arctica / Image courtesy: FEI Company
|Microscopy||Model: FEI TALOS ARCTICA|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 73000 X / Nominal defocus max: 16000 nm / Nominal defocus min: 5000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE|
|Specimen holder||Cryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Image recording||Average exposure time: 11 sec. / Electron dose: 69 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 1673|
|Image scans||Sampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 44 / Used frames/image: 1-44|
|Software||Name: PHENIX / Version: 1.11.1_2580: / Classification: refinement|
|Image processing||Details: Counting mode, 250 ms frames, exposure rate of ~1.95 e- pixel-1 s-1, total exposure of ~69 e- angstrom-2 (1.57 e- angstrom-2 frame-1).|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Particle selection||Num. of particles selected: 1232543 / Details: Difference-of-Gaussian-picked.|
|Symmetry||Point symmetry: C2 (2 fold cyclic)|
|3D reconstruction||Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24308 / Symmetry type: POINT|
|Atomic model building||B value: 67 / Protocol: FLEXIBLE FIT / Space: REAL|
|Atomic model building||PDB-ID: 4N7P|
Pdb chain-ID: A
|Refine LS restraints|
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