12.2 Nucleic acid and virus crystallography

Nucleic acid crystallography uncovers the intricate structures of DNA and RNA, revealing their roles in biological processes. Challenges like flexibility and weak diffraction are overcome with advanced techniques, providing insights crucial for understanding gene regulation and developing therapies.

Virus crystallography combines X-ray methods with cryo-EM to tackle large, complex structures. These studies unveil viral architecture, assembly mechanisms, and host interactions, paving the way for antiviral drug design and vaccine development.

Challenges of Nucleic Acid Crystallography

Structural Complexities and Experimental Considerations

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• High flexibility and conformational variability of DNA and RNA molecules present unique challenges
• Negatively charged phosphate backbone requires careful crystal packing and solvent condition management
• Weak diffraction patterns typically observed necessitate use of synchrotron radiation sources
• Cryocrystallography techniques minimize radiation damage and improve data quality
  • Involves flash-freezing crystals in liquid nitrogen
  • Reduces thermal motion and extends crystal lifetime during data collection
• Heavy atom derivatization techniques solve the phase problem
  • Osmium tetroxide modification
  • Bromination of thymine bases (5-bromouracil)
  • Allows determination of initial phase information

Advanced Methods for Structure Determination

• Molecular replacement employs known structural motifs as search models
  • A-form or B-form helices serve as common starting points
  • Helps determine initial phases for unknown structures
• Refinement techniques crucial for accurate structure determination
  • Stereochemical restraints maintain proper bond lengths and angles
  • Non-crystallographic symmetry improves electron density maps
• Advanced synchrotron beamlines enable high-resolution data collection
  • Microfocus beamlines allow use of smaller crystals
  • Increased flux and improved detectors enhance data quality

Importance of Nucleic Acid Structures

Fundamental Biological Processes

• Provide crucial insights into DNA replication, transcription, and translation mechanisms
  • Reveal enzyme binding sites and catalytic centers
  • Elucidate structural changes during these processes
• Structural information on DNA-protein and RNA-protein complexes illuminates gene regulation
  • Transcription factor binding sites (TATA box, enhancers)
  • Ribosome structure and function in translation
• Three-dimensional structures of ribozymes and aptamers reveal catalytic and ligand-binding capabilities
  • Group I introns demonstrate RNA's catalytic potential
  • Aptamers (theophylline-binding RNA) showcase specific molecular recognition

Medical and Therapeutic Applications

• Essential for rational drug design targeting specific DNA or RNA sequences
  • Anticancer drugs (cisplatin, doxorubicin)
  • Antiviral therapies (HIV reverse transcriptase inhibitors)
• Structural basis of DNA damage and repair mechanisms advances cancer research
  • Nucleotide excision repair complexes
  • Base excision repair enzymes
• RNA structure determination contributes to RNA-based therapeutics development
  • siRNA design for gene silencing
  • mRNA vaccine optimization (COVID-19 vaccines)
• Telomere architecture insights play a role in aging research
  • G-quadruplex structures in telomeric DNA
  • Telomerase enzyme structure and function

Methods in Virus Crystallography

Integrated Structural Biology Approaches

• Cryo-electron microscopy (cryo-EM) combined with X-ray crystallography overcomes challenges of large viral particles
  • Cryo-EM provides overall shape and symmetry information
  • X-ray crystallography offers high-resolution atomic details
• Single-particle analysis reconstructs 3D virus structures from 2D cryo-EM images
  • Involves aligning and averaging thousands of particle images
  • Utilizes symmetry to improve resolution
• Hybrid methods elucidate complete virus structures
  • Integrates X-ray crystallography, cryo-EM, and computational modeling
  • Reveals both protein and nucleic acid components

Advanced Crystallographic Techniques

• Phase extension methods improve resolution of virus structures
  • Non-crystallographic symmetry averaging exploits viral symmetry
  • Molecular replacement using known structural components
• Time-resolved crystallography studies dynamic processes
  • Captures viral assembly and maturation intermediates
  • Utilizes pump-probe experiments with laser excitation
• Micro-electron diffraction (MicroED) determines high-resolution structures from nanocrystals
  • Enables study of small viral proteins or fragments
  • Overcomes size limitations of traditional crystallography
• X-ray free-electron lasers (XFELs) facilitate native state studies
  • Serial femtosecond crystallography captures diffraction before radiation damage
  • Allows room-temperature data collection on sensitive viral samples

Structural Features of Nucleic Acids and Viruses

Nucleic Acid Structural Insights

• DNA helical structure revealed through crystallography
  • Major and minor grooves (potential drug binding sites)
  • Base stacking and hydrogen bonding patterns
  • Various conformations: A-DNA, B-DNA, Z-DNA
• RNA structures exhibit complex tertiary interactions
  • Pseudoknots (telomerase RNA)
  • Kissing loops (HIV dimerization initiation site)
  • Ribose zippers (group I intron)

Viral Architecture and Function

• Viral capsid structures provide insights into assembly and stability
  • Icosahedral symmetry (adenovirus)
  • Helical arrangement (tobacco mosaic virus)
• Genomic material arrangement within viral particles elucidated
  • Ordering of nucleic acids in helical viruses (influenza)
  • Packaging of dsDNA in bacteriophages (phi29)
• Virus-receptor complexes reveal molecular basis of host specificity
  • Influenza hemagglutinin binding to sialic acid
  • HIV gp120 interaction with CD4 receptor
• Conformational changes in viral proteins observed
  • Fusion proteins (influenza hemagglutinin, HIV gp41)
  • Genome packaging motors (bacteriophage portal proteins)
• Comparative analysis highlights conserved structural motifs
  • Jelly-roll fold in many viral capsid proteins
  • Evolutionary relationships between virus families (picornavirus superfamily)