MODULE 4: Chapter 5.3 Flashcards
Who first modeled the structure of DNA using X-ray diffraction data?
James Watson and Francis Crick
They used data from Rosalind Franklin.
In what year was the first protein molecular structure solved by X-ray crystallography?
1957
Which protein’s low-resolution structure was reported by John Kendrew?
Myoglobin
Who shared the 1962 Nobel Prize in Chemistry with John Kendrew?
Max Perutz
What are the three primary methods for determining the molecular structure of proteins?
- X-ray crystallography
- Nuclear magnetic resonance (NMR) spectroscopy
- Cryo-electron microscopy
What can insights from a protein’s structure help us understand?
- Normal cellular function
- Contribution of mutant proteins to disease
- Drug design to disrupt protein function
What principle does X-ray crystallography rely on?
Diffraction of X-rays by a protein crystal
What is formed from the measurements of scattered X-rays in X-ray crystallography?
An electron density map
What is the significance of the electron density map in protein modeling?
It helps to build a model of the protein that matches the regions of electron density.
What are the two most difficult steps in X-ray crystallography?
- Growing diffraction-quality crystals
- Determining the phases of the diffracted X-rays
What is required for protein crystallization?
High concentrations of proteins
What is a heavy atom derivative used for in X-ray crystallography?
To determine the phases of the diffracted X-rays
What does the resolution of a protein structure indicate?
The level of detail in the electron density maps
What style of protein modeling was developed by Jane Richardson?
Ribbon style
What does NMR spectroscopy utilize to determine the structure of proteins?
Intrinsic magnetic properties of certain atoms
What isotopes are most commonly used in NMR spectroscopy?
- 1H
- 15N
- 13C
What is the limitation of protein size for NMR structure determination?
Proteins must be less than ∼100 kDa
What type of data does NMR spectroscopy provide?
Relative locations of atoms
What type of structures can NMR data result in?
A family of structures
What was a significant advancement in cryo-electron microscopy recognized by the Nobel Prize?
Development of cryo-EM as a tool for biomolecular structure determination
What is the advantage of cryo-electron microscopy over X-ray crystallography?
No need for crystals or high sample concentrations
What is commonly done to biological samples before imaging in cryo-EM?
Rapidly frozen to produce vitreous ice
What is a key feature of the images obtained from cryo-electron microscopy?
They are two-dimensional projections of three-dimensional particles
What is the purpose of computational algorithms in cryo-EM?
To create three-dimensional images from two-dimensional projections
What technique uses an electron beam focused by electromagnetic lenses to produce two-dimensional projections of three-dimensional objects?
Cryo-electron microscopy (cryo-EM)
Cryo-EM is used to analyze large assemblies and multiple conformations.
What is required to create high-resolution two-dimensional projections in cryo-electron microscopy?
Computational algorithms to identify similarly oriented objects
These algorithms help in generating high-resolution images from randomly oriented samples.
What are the primary types of protein samples used in X-ray crystallography and NMR spectroscopy?
X-ray crystallography uses ordered arrays of protein molecules in a crystal; NMR spectroscopy uses concentrated protein solutions
Each method has its own limitations based on the type of sample used.
What is a significant limitation of X-ray crystallography?
Difficulties in obtaining protein crystals for new proteins
Conditions for crystallization are often unknown and challenging.
What type of protein regions are especially difficult to analyze using X-ray crystallography?
Disordered regions or proteins in multiple conformations
These cannot pack in an ordered, regular pattern needed for crystal formation.
What is the theoretical size limitation of proteins studied by X-ray crystallography?
There is no theoretical size limitation
This allows for the study of very large proteins.
What concentration range is typically required for NMR spectroscopy?
Approximately 0.1 to 0.5 mM
This concentration may not be achievable for all proteins.
What is a major challenge in determining structures of large proteins using NMR spectroscopy?
Signal loss from larger proteins due to slower reorientation
This limits full structure determinations to relatively small proteins and complexes.
What advantage does NMR spectroscopy have over X-ray crystallography?
Ability to observe conformational changes or dynamic fluctuations
This is particularly useful for studying proteins that undergo structural changes.
Fill in the blank: A structure determination technique where the diffraction of X-rays from crystals of biomolecules is used to determine the locations of atoms in the structure is called _______.
X-ray crystallography
Fill in the blank: A method of structure determination that detects nuclear spin properties of certain atoms (1H, 13C, 15N) to deduce their relative locations is known as _______.
NMR spectroscopy
What is molecular replacement in the context of X-ray crystallography?
A procedure comparing the phases of diffracted X-rays with those from a similar protein of known structure
This technique helps in solving the phase problem in crystallography.
True or False: Cryo-electron microscopy can visualize biomolecular structures using focused beams of electrons.
True
