CRYSTALLOGRAPHY AND ION CHANNELS

What is the main purpose of X-ray crystallography?

To determine the 3D atomic structure of proteins by analyzing how X-rays scatter through a crystal.

Why must a protein be crystallized for X-ray crystallography?

Crystals provide a repeating, ordered structure that allows X-rays to diffract and generate interpretable patterns.

What is vapor diffusion in protein crystallization?

A method where water vapor equilibrates between protein and precipitant solutions, increasing protein concentration to promote crystallization.

Why are X-rays used instead of visible light in crystallography?

X-rays have a wavelength similar to atomic distances, allowing resolution at the atomic scale.

What is a structure factor (Fhkl)?

A mathematical description of a diffraction spot, with amplitude (measured) and phase (lost).

What is the phase problem in crystallography?

The inability to directly measure the phase of diffracted X-rays, which is required to generate an electron density map.

How does Multiple Isomorphous Replacement (MIR) solve the phase problem?

By soaking crystals with heavy atoms, then comparing diffraction to native crystals to estimate phase data.

What is Single/Multi-wavelength Anomalous Dispersion (SAD/MAD)?

A technique using atoms that scatter X-rays differently at specific wavelengths to estimate phases.

What is Molecular Replacement (MR)?

A method that uses a previously solved similar protein structure to estimate phases.

What is an electron density map?

A 3D representation of electron cloud positions used to build the atomic model of a protein.

What are Rwork and Rfree in crystallography?

Rwork measures model agreement with 90% of data; Rfree uses 10% withheld data to prevent bias.

What is a good range for Rwork and Rfree?

They should be low and within ~5% of each other to indicate a reliable model.

What are other ways to validate a crystallography model?

Ramachandran plot, bond angles, omit maps, and agreement with biochemical data.

What are limitations of X-ray crystallography?

Requires crystallizable, stable proteins; does not show dynamics; difficult to detect protons.

What are voltage-gated ion channels?

Membrane proteins that open/close in response to changes in membrane potential, allowing ion flow.

What do Naᵥ channels do?

They open to allow Na⁺ influx during depolarization, initiating action potentials.

What is the structure of a voltage-gated sodium channel?

Four domains (I–IV), each with six transmembrane helices (S1–S6), with S4 as voltage sensor.

What is the role of the selectivity filter (SF) in Naᵥ channels?

Determines ion specificity using charged residues like DEKA to select for Na⁺ over K⁺.

How do bacterial sodium channels differ from eukaryotic ones?

They are smaller, symmetrical, and have a simpler selectivity filter (EEEE).

What are voltage-gated potassium (Kv) channels?

Tetrameric channels that open upon depolarization to allow K⁺ out, aiding repolarization.

What is the difference between delayed rectifier and transient Kv channels?

Delayed rectifiers stay open longer; transient channels inactivate quickly.

What happens during depolarization in an action potential?

Naᵥ channels open, Na⁺ enters the cell.

What happens during repolarization in an action potential?

Kv channels open, K⁺ exits the cell.

What are TRP channels?

Ion channels involved in sensory perception, activated by temperature, chemicals, and other stimuli.

What do TRP channels do when activated?

They open to allow ion flow, leading to depolarization and triggering nerve impulses.

Where is KCNQ1 expressed, and what does it do?

In heart, inner ear, and epithelial cells; forms Kv7.1 channels that maintain ion balance and cardiac rhythm.

What protein forms a functional potassium channel with KCNQ1?

KCNE1, a beta subunit that regulates KCNQ1 channel activity.

What is Long QT syndrome?

A condition caused by KCNQ1 mutations that delays cardiac repolarization, risking arrhythmias and sudden death.

What is Jervell and Lange-Nielsen syndrome?

A form of Long QT syndrome that also causes hearing loss, linked to KCNQ1 mutations.

What are the three models of voltage sensing in VGCs?

Sliding Helix Model, Transporter Model, and KvAP (Paddle) Model.

Describe the Sliding Helix Model.

Voltage sensor (S4) moves through membrane, forming sequential interactions with neighboring residues.

Describe the Transporter Model.

Voltage causes a reshaped electrical field that shifts sensor domains to open the channel.

Describe the KvAP Model.

A helical paddle (S3-S4) moves across the membrane like an oar, activating the channel.