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- PDB-6nbd: Human methemoglobin state 2 determined using single-particle cryo... -
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Open data
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Basic information
Entry | Database: PDB / ID: 6nbd | ||||||
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Title | Human methemoglobin state 2 determined using single-particle cryo-EM at 200 keV | ||||||
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![]() | OXYGEN TRANSPORT / heme-binding / hetero-4-mer / globin | ||||||
Function / homology | ![]() nitric oxide transport / cellular oxidant detoxification / hemoglobin binding / hemoglobin alpha binding / haptoglobin-hemoglobin complex / organic acid binding / renal absorption / hemoglobin complex / oxygen transport / Scavenging of heme from plasma ...nitric oxide transport / cellular oxidant detoxification / hemoglobin binding / hemoglobin alpha binding / haptoglobin-hemoglobin complex / organic acid binding / renal absorption / hemoglobin complex / oxygen transport / Scavenging of heme from plasma / endocytic vesicle lumen / blood vessel diameter maintenance / hydrogen peroxide catabolic process / oxygen carrier activity / regulation of blood pressure / Late endosomal microautophagy / Heme signaling / carbon dioxide transport / Erythrocytes take up oxygen and release carbon dioxide / response to hydrogen peroxide / Erythrocytes take up carbon dioxide and release oxygen / Cytoprotection by HMOX1 / platelet aggregation / oxygen binding / Chaperone Mediated Autophagy / positive regulation of nitric oxide biosynthetic process / Factors involved in megakaryocyte development and platelet production / tertiary granule lumen / ficolin-1-rich granule lumen / blood microparticle / iron ion binding / heme binding / Neutrophil degranulation / extracellular space / extracellular exosome / extracellular region / membrane / metal ion binding / cytosol Similarity search - Function | ||||||
Biological species | ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | ||||||
![]() | Herzik Jr., M.A. / Wu, M. / Lander, G.C. | ||||||
Funding support | ![]()
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![]() | ![]() 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. | ||||||
History |
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 802.4 KB | Display | ![]() |
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PDB format | ![]() | 704.1 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 1.7 MB | Display | ![]() |
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Full document | ![]() | 1.8 MB | Display | |
Data in XML | ![]() | 114.4 KB | Display | |
Data in CIF | ![]() | 154 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 0408MC ![]() 0406C ![]() 0407C ![]() 0409C ![]() 6nbbC ![]() 6nbcC M: map data used to model this data C: citing same article ( |
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Similar structure data | |
EM raw data | ![]() Data size: 1.9 TB Data #1: Raw, unaligned movie stacks of human methemoglobin acquired on a Talos Arctica using a K2 direct electron detector [micrographs - multiframe]) |
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Links
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Assembly
Deposited unit | ![]()
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Number of models | 9 |
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Components
#1: Protein | Mass: 14993.159 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() Plasmid details: Obtained in lyophilized form from Sigma Aldrich References: UniProt: P69905 #2: Protein | Mass: 15459.697 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() Plasmid details: Obtained in lyophilized from from Sigma Aldrich References: UniProt: P68871 #3: Chemical | ChemComp-HEM / |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Human methemoglobin / Type: COMPLEX Details: Lyophilized human methemoglobin was obtained from Sigma Aldrich Entity ID: #1-#2 / Source: NATURAL | |||||||||||||||||||||||||
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Molecular weight | Value: 0.064 MDa / Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: ![]() | |||||||||||||||||||||||||
Buffer solution | pH: 7.4 | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Lyophilized human methemoglobin (Sigma Aldrich) was solubilized in PBS pH 7.4. | |||||||||||||||||||||||||
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. |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / 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 / Specimen holder 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: 1 / Num. of real images: 1673 |
Image scans | Sampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 44 / Used frames/image: 1-44 |
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Processing
Software | Name: PHENIX / Version: 1.11.1_2580: / Classification: refinement | ||||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: Counting mode, 250 ms frames, exposure rate of ~1.95 e-/pix/sec, total exposure of ~69 e-/angstrom-2 (1.57 e-/angstrom-2/frame). | ||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1615738 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11501 / Algorithm: BACK PROJECTION / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 93 / Protocol: FLEXIBLE FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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