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Questions and Answers
What is a primary characteristic of Single Particle Analysis in cryo-electron microscopy?
What is a primary characteristic of Single Particle Analysis in cryo-electron microscopy?
Cryo-tomography typically yields high-resolution data.
Cryo-tomography typically yields high-resolution data.
False
What is the main purpose of averaging in cryo-electron microscopy?
What is the main purpose of averaging in cryo-electron microscopy?
To increase signal and decrease noise.
In Cryo-tomography, tilt-series are collected at a single __________.
In Cryo-tomography, tilt-series are collected at a single __________.
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Match the following terms with their corresponding descriptions:
Match the following terms with their corresponding descriptions:
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What is necessary to obtain the 3D structure of a sample particle in cryoEM?
What is necessary to obtain the 3D structure of a sample particle in cryoEM?
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In cryoEM, image processing is computationally inexpensive and quick.
In cryoEM, image processing is computationally inexpensive and quick.
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What term describes the 2D projection of a 3D object in electron microscopy?
What term describes the 2D projection of a 3D object in electron microscopy?
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Sine waves can be represented in both 'real-space' and _________ space.
Sine waves can be represented in both 'real-space' and _________ space.
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Match the following cryoEM techniques or concepts with their descriptions:
Match the following cryoEM techniques or concepts with their descriptions:
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What is the main purpose of Fourier Shell Correlation (FSC) in cryoEM?
What is the main purpose of Fourier Shell Correlation (FSC) in cryoEM?
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In Cryo-electron Tomography, samples are always purified before imaging.
In Cryo-electron Tomography, samples are always purified before imaging.
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What is the primary technique used to achieve molecular resolution in cellular cryoET?
What is the primary technique used to achieve molecular resolution in cellular cryoET?
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Cryo-tomography involves collecting ___ images at different orientations to create a 3D structure.
Cryo-tomography involves collecting ___ images at different orientations to create a 3D structure.
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Match the cryo-analysis techniques with their descriptions:
Match the cryo-analysis techniques with their descriptions:
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Which statement correctly describes a limitation of cryoET?
Which statement correctly describes a limitation of cryoET?
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Subtomogram averaging results in lower resolution images than single particle analysis.
Subtomogram averaging results in lower resolution images than single particle analysis.
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What type of images are collected in single particle analysis?
What type of images are collected in single particle analysis?
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Study Notes
CryoEM - 2 Main Flavors
- CryoEM is a technique for imaging biological materials at extremely low temperatures
- There are two main types of cryoEM: Single Particle Analysis and Cryo-Tomography
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Single Particle Analysis
- The sample can be purified or not
- Thousands of images are taken at different locations
- Orientations are determined to align and average 2D projections
- The 2D projections are used to “build” the original 3D structure
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Cryo-Tomography:
- The sample can be purified or not
- Tilt-series are collected at a single location
- Tilt-series are back-projected into tomogram (3D)
- 3D Subtomograms are aligned and averaged
- Subtomogram averaging provides high resolution
The Projection Theorem
- The projection theorem is a fundamental concept in image processing
- The projection theorem explains how a 3D structure is projected in a 2D image
- The Fourier transform of a projection image is equivalent to a slice of the 3D Fourier transform
- The information lost in a 2D projection can be recovered by performing multiple projections
- 2D projections are used to “build” the original 3D structure
Resolution in CryoEM
- Resolution is a crucial parameter in cryoEM that determines the level of detail that can be observed
- The resolution is estimated and not measured directly
- There are different ways to estimate the resolution
- The resolution is limited by the wavelength of the electron beam and the sampling at the camera level (pixel size)
- Information about the resolution is crucial for interpreting cryoEM data
Automated Modeling and Validation of Protein Complexes in Cryo-EM Maps
- Automated modeling and validation of protein complexes in cryoEM maps is an active area of research
- New methods and tools are being developed to address the challenges of building accurate 3D models from cryoEM maps
- These methods leverage both computational and experimental data
Fourier Shell Correlation (FSC)
- FSC is a measure of self-consistency of the data and the reconstruction process
- FSC is used to assess the resolution of a cryoEM reconstruction
- FSC is a function of spatial frequency or resolution
- FSC is calculated by comparing the Fourier transforms of two independent reconstructions
- Low-resolution (lower spatial frequency) information is more consistent than high-resolution (higher spatial frequency) information
Cryoelectron Tomography (CryoET)
- CryoET is a powerful technique for imaging biological samples in their native state
- CryoET can provide 3D structural information about complex cellular structures
- CryoET is used to study the organization and function of biological processes
- CryoET provides information about the location and orientation of proteins and other molecules within cellular structures
Cellular CryoET
- Cellular cryoET is a powerful tool for studying the structure and function of cellular structures
- Cellular CryoET enables the visualization of complex cellular processes in their native environment
- Cellular cryoET is complementary to other imaging techniques such as light microscopy
CryoEM: seeing is believing
- Subtomogram averaging is used to increase the resolution of cryoET reconstructions
- Subtomogram averaging can achieve near-atomic resolution for individual protein structures
- Subtomogram averaging allows for the study of biological processes at a molecular level
“Projection” imaging in EM
- One EM image is not sufficient to deduce the structure of a sample particle
- Multiple images are required to reconstruct the 3D structure of a sample
- Images are taken at different orientations of the sample
- The images are then aligned and averaged to produce a 3D reconstruction of the sample
CryoEM Images: Low S/N
- CryoEM images have very low signal-to-noise ratio (S/N)
- The low S/N is due to the use of low electron dose to minimize radiation damage
- The low S/N also arises from shot noise and aberrations in the imaging system
- Image processing is crucial to improve the S/N of cryoEM images and extract meaningful information
Image processing
- Image processing is an essential step in cryoEM data analysis
- Images are transformed and processed to enhance their signal and reduce the noise
- Image transformations are performed in Fourier space, which is the space of spatial frequencies
- FFT are also powerful tools for identifying issues with data collection in cryoEM
- The conversion from real space to FFT is lossless
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Description
This quiz covers the two main flavors of cryo-electron microscopy (CryoEM): Single Particle Analysis and Cryo-Tomography. Additionally, it delves into the projection theorem, a key concept in image processing that describes the projection of 3D structures into 2D images. Test your knowledge on these advanced imaging techniques and their principles!
