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- PDB-6wxb: Cryo-EM Structure of Influenza Hemagglutinin (HA) Trimer Vitrifie... -
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Open data
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Basic information
Entry | Database: PDB / ID: 6wxb | ||||||||||||
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Title | Cryo-EM Structure of Influenza Hemagglutinin (HA) Trimer Vitrified Using Back-it-up | ||||||||||||
![]() | Hemagglutinin | ||||||||||||
![]() | VIRAL PROTEIN / Hemagglutinin / trimer / Influenza / back-it-up / through-grid wicking | ||||||||||||
Function / homology | ![]() viral budding from plasma membrane / clathrin-dependent endocytosis of virus by host cell / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane / virion membrane / membrane Similarity search - Function | ||||||||||||
Biological species | ![]() ![]() | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å | ||||||||||||
![]() | Tan, Y.Z. / Rubinstein, J.L. | ||||||||||||
Funding support | ![]()
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![]() | ![]() Title: Through-grid wicking enables high-speed cryoEM specimen preparation. Authors: Yong Zi Tan / John L Rubinstein / ![]() Abstract: Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air-water interface. Here, ...Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air-water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass-fiber filter held against the opposite side, often called the `back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic specimen application and through-grid wicking were combined in a high-speed specimen-preparation device that was named `Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations. | ||||||||||||
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 | ![]() | 471.3 KB | Display | ![]() |
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PDB format | ![]() | 408.4 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 21954MC ![]() 6wx6C M: map data used to model this data C: citing same article ( |
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Similar structure data | |
EM raw data | ![]() Data size: 1.2 TB Data #1: Unaligned Falcon IV movies [micrographs - multiframe] Data #2: Aligned micrographs [micrographs - single frame] Data #3: Final Refined Particles with Euler Angles and Shifts [picked particles - single frame - processed]) |
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Links
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Assembly
Deposited unit | ![]()
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1 |
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Components
#1: Protein | Mass: 62855.215 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Strain: A/Hong Kong/1/1968 H3N2 / Gene: HA / Production host: ![]() #2: Sugar | ChemComp-NAG / #3: Sugar | Has ligand of interest | N | |
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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: Influenza Hemagglutinin Trimer (H3N2) (A/Hong Kong/1/1968) Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT |
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Molecular weight | Value: 0.19 MDa / Experimental value: NO |
Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() |
Buffer solution | pH: 7.4 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: Grid was glow discharged on both sides for 120s each. Grid material: COPPER/RHODIUM / Grid type: Homemade |
Vitrification | Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE-PROPANE / Humidity: 50 % / Chamber temperature: 298 K Details: Back-it-up (ultrasonic specimen application and through-grid wicking in a high-speed specimen preparation device) was used |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Cs: 2.7 mm / Alignment procedure: ZEMLIN TABLEAU |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 9 sec. / Electron dose: 45 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1556 |
Image scans | Sampling size: 14 µm / Width: 4096 / Height: 4096 |
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Processing
Software |
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 378143 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C3 (3 fold cyclic) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128305 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 3WHE | ||||||||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE / Stereochemistry target values: CDL v1.2 | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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