Protein Structure Determination by X-ray Crystallography

What is the main goal of X-ray crystallography in protein studies?

To determine the atomic structure of proteins.

Name two applications of protein structure determination by X-ray crystallography.

Drug design and understanding binding mechanisms.

List one advantage of using X-ray crystallography.

High-resolution structures.

What temperature is X-ray crystallography typically performed at?

−173 °C.

What is a disadvantage of the structure obtained from X-ray crystallography?

It provides a static, average structure.

What type of proteins are difficult to crystallize?

Membrane proteins.

What is one alternative method to X-ray crystallography?

Nuclear Magnetic Resonance (NMR).

What are the challenges associated with crystallization?

No universal rules; requires pure, concentrated, stable protein; not all proteins crystallize.

What is Bragg’s Law related to in X-ray crystallography?

It explains constructive interference when path difference equals nλ.

What does flash freezing with cryoprotectant help achieve in crystallography?

It reduces radiation damage and traps enzyme intermediates.

What type of X-ray source is characterized by low brightness and a fixed wavelength?

Laboratory X-ray source.

What is the function of XFELs in protein crystallography?

They capture ultrafast events by streaming microcrystals.

In diffraction analysis, what do X-rays diffract off?

Electron clouds.

How is the resolution in X-ray crystallography measured?

In Ångströms (1 Å = 0.1 nm).

What does a lower Ångström value indicate about the resolution?

Higher resolution.

What is the phase problem in X-ray crystallography?

X-ray measures amplitude but not phase, which must be estimated to build a model.

Name one method used to solve the phase problem.

Molecular replacement.

What must be validated when building a model in X-ray crystallography?

Bond lengths/angles and side-chain rotamers.

What is the significance of the Ramachandran plot in structure validation?

It evaluates the φ and ψ angles of amino acids.

What was the application of X-ray crystallography in the context of SARS-CoV-2?

To determine the structure of the spike protein for vaccine design.

What does a hanging drop technique in crystallization provide?

Good visibility and control.

What is one reason cryo-EM is advantageous in protein studies?

It is great for large complexes.

What is the role of radio waves in NMR?

They are used to probe the protein structure.

What are typical conditions for crystallization variable adjustments?

pH, buffer, salt types/concentration, temperature, and protein concentration.

Which technique is suitable for automation and high throughput in protein crystallization?

Sitting drop technique.

What does the B-factor in model validation refer to?

The temperature factor of atoms in the model.

Why is hydrogen often not visible in X-ray crystallography results?

X-rays detect electrons poorly, making hydrogens hard to visualize.

What are the axes used to define a unit cell in a crystal structure?

Three axes: a, b, c.

What characterizes a synchrotron X-ray source compared to a laboratory source?

It is tunable, faster, and has higher brightness.

What does data collection involve in crystallography?

Recording X-ray diffraction patterns to build a 3D dataset.

What are microcrystals used for in SFX (Serial Femtosecond Crystallography)?

To capture ultrafast events in protein dynamics.