NMR and X-ray crystallography in structure prediction MCQs With Answer

NMR and X‑ray crystallography are cornerstone techniques in structure prediction and rational drug design. This quiz collection targets M.Pharm students preparing for advanced coursework and exams in Principles of Drug Discovery. The questions cover theoretical principles, experimental workflows, data interpretation, strengths and limitations of each method, and how structural information guides ligand design, fragment screening, and SAR. Expect items on pulse sequences, NOE, 2D/3D NMR experiments, sample requirements, phasing methods, electron density maps, resolution, refinement statistics, and practical challenges like twinning or flexible regions. Working through these MCQs will reinforce conceptual understanding and practical decision‑making for applying NMR and crystallography in drug discovery.

Q1. Which NMR phenomenon provides direct distance restraints between hydrogen atoms and is widely used to determine three‑dimensional structures of small to medium‑sized molecules?

• Nuclear Overhauser Effect (NOE)
• Scalar coupling (J‑coupling)
• Chemical shift anisotropy
• Exchange broadening

Correct Answer: Nuclear Overhauser Effect (NOE)

Q2. In X‑ray crystallography, which parameter indicates the overall agreement between observed and calculated structure factors and is commonly used to judge model quality during refinement?

• R‑factor (Rwork)
• B‑factor (temperature factor)
• Resolution shell
• Unit cell volume

Correct Answer: R‑factor (Rwork)

Q3. Which phasing method exploits differences in scattering due to incorporation of heavy atoms or anomalous scatterers to obtain initial phase information?

• Multiple Isomorphous Replacement (MIR) / Anomalous methods (MAD/SAD)
• Molecular Replacement (MR)
• Direct methods
• Refinement by simulated annealing

Correct Answer: Multiple Isomorphous Replacement (MIR) / Anomalous methods (MAD/SAD)

Q4. Which 2D NMR experiment correlates proton and directly bonded heteronuclei (commonly 1H‑13C or 1H‑15N) and is essential for assignment of backbones in biomolecules?

• HSQC (Heteronuclear Single Quantum Coherence)
• NOESY
• DEPT
• TOCSY

Correct Answer: HSQC (Heteronuclear Single Quantum Coherence)

Q5. What is the typical practical resolution threshold (in Å) below which side chain orientations and ligand conformations are reliably interpreted from X‑ray electron density maps?

• Approximately 2.5 Å or better (≤ 2.5 Å)
• Approximately 4.0 Å
• Approximately 6.0 Å
• Resolution is irrelevant for interpretation

Correct Answer: Approximately 2.5 Å or better (≤ 2.5 Å)

Q6. Which NMR‑based screening method is specifically designed to detect weak ligand binding to proteins by observing ligand signal intensity changes in the presence of protein?

• STD‑NMR (Saturation Transfer Difference NMR)
• NOESY
• HMQC
• Diffusion ordered spectroscopy (DOSY)

Correct Answer: STD‑NMR (Saturation Transfer Difference NMR)

Q7. In X‑ray crystallography, what problem arises when two or more crystalline domains are present in different orientations, complicating data integration and map interpretation?

• Twinning
• Radiation damage
• Poor cryo‑protection
• High mosaic spread

Correct Answer: Twinning

Q8. Which of the following NMR observables is most directly sensitive to electronic environment and often used to map ligand binding sites through chemical shift perturbation?

• Chemical shift
• NOE cross‑peak intensity
• Scalar coupling constant
• Spin‑lattice relaxation time (T1)

Correct Answer: Chemical shift

Q9. Which step is unique to X‑ray crystallography sample preparation compared with solution NMR for structural studies of proteins?

• Growing well‑ordered single crystals of the protein or protein–ligand complex
• Isotopic labeling with 13C/15N
• Recording 2D heteronuclear spectra
• Measurement of relaxation rates

Correct Answer: Growing well‑ordered single crystals of the protein or protein–ligand complex

Q10. Which refinement statistic describes the level of thermal motion or static disorder of atoms in an X‑ray structure?

• B‑factor (atomic displacement parameter)
• Rfree
• Completeness
• Multiplicity

Correct Answer: B‑factor (atomic displacement parameter)

Q11. Which NMR technique provides through‑bond correlations useful for establishing carbon skeleton connectivity in small organic molecules?

• HMBC (Heteronuclear Multiple Bond Correlation)
• NOESY
• CPMAS
• EXSY

Correct Answer: HMBC (Heteronuclear Multiple Bond Correlation)

Q12. In molecular replacement (MR) for X‑ray structure solution, what is required as the starting point to obtain phases?

• An existing related model (homologous structure) to position into the unit cell
• Incorporation of heavy atoms into crystals
• Recording anomalous diffraction at multiple wavelengths
• Direct interpretation of noisy electron density

Correct Answer: An existing related model (homologous structure) to position into the unit cell

Q13. Which NMR parameter is commonly used to detect conformational exchange on microsecond to millisecond timescales and can indicate flexible regions in proteins?

• R2 relaxation dispersion
• NOE buildup curves
• Scalar coupling JHN‑HA
• Diffusion coefficient

Correct Answer: R2 relaxation dispersion

Q14. Which strategy in crystallography helps reduce radiation damage and improve data quality during X‑ray data collection?

• Cryo‑cooling crystals to cryogenic temperatures (e.g., 100 K)
• Increasing exposure time per frame
• Avoiding cryo‑protectants entirely
• Collecting at room temperature only

Correct Answer: Cryo‑cooling crystals to cryogenic temperatures (e.g., 100 K)

Q15. Which experimental NMR method can measure residual dipolar couplings (RDCs) to provide long‑range angular restraints useful for improving global fold accuracy?

• Alignment media experiments that measure RDCs (e.g., Pf1, bicelles)
• Standard 1D proton NMR in isotropic solution
• Static solid‑state NMR without alignment
• NOESY alone

Correct Answer: Alignment media experiments that measure RDCs (e.g., Pf1, bicelles)

Q16. Which map type in crystallography highlights peaks corresponding to differences between observed and model‑calculated electron density and is useful for locating ligands and ions?

• Fo − Fc difference map
• 2Fo − Fc map
• Patterson map
• Unit cell electron density map

Correct Answer: Fo − Fc difference map

Q17. Which limitation is most associated with using solution NMR for structural studies of large proteins (>30–40 kDa) without specialized approaches?

• Severe signal broadening and overlap due to slow tumbling
• Inability to detect hydrogen atoms
• Requirement for crystallization
• Complete insensitivity to ligand binding

Correct Answer: Severe signal broadening and overlap due to slow tumbling

Q18. In fragment‑based drug discovery, which structural technique combination is often used to identify and rapidly optimize weakly binding fragments?

• Biophysical screening (NMR/SPR) combined with X‑ray co‑crystallography
• Only high‑throughput biochemical assays
• Mass spectrometry imaging alone
• Genome sequencing of target organisms

Correct Answer: Biophysical screening (NMR/SPR) combined with X‑ray co‑crystallography

Q19. Which refinement target helps prevent overfitting by assessing how well a model predicts a subset of reflections not used during refinement?

• Rfree
• Rwork
• Completeness
• Wilson B‑factor

Correct Answer: Rfree

Q20. Which crystallographic concept defines the smallest repeating unit in a crystal and, together with symmetry operations, generates the whole lattice?

• Unit cell
• Asymmetric unit
• Molecular envelope
• Electron density blob

Correct Answer: Unit cell

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