Difficulty: Intermediate | Prerequisites: Part 1 of these notes (early research, DNA as the genetic material), basic chemistry (hydrogen bonds, molecular structure).
Once scientists established that DNA was the genetic material, the next question was: what does the molecule look like, and how does its shape explain heredity? Several teams raced to answer this using X-ray crystallography and physical model-building. This topic covers the technique of X-ray crystallography, the key contributions of Rosalind Franklin and Maurice Wilkins, the failed triple-helix model, and the breakthroughs that led Watson and Crick to the correct double-helix structure. Understanding these events is essential for grasping how structure-function relationships work at the molecular level.
X-ray crystallography let scientists "see" DNA's shape by bouncing X-rays off molecules and reading the resulting diffraction patterns. Rosalind Franklin's Photo 51 showed the characteristic X-pattern of a helix and revealed critical dimensions. Watson and Crick combined this data with Chargaff's base-pairing rules and corrected chemical structures to build the double-helix model: two antiparallel sugar-phosphate backbones on the outside, with complementary base pairs (A-T, G-C) on the inside held together by hydrogen bonds.
X-ray crystallography
A technique for determining molecular structure by bombarding a sample with X-rays and analysing the resulting diffraction pattern. In simple terms, it is a way to "photograph" the arrangement of atoms in a molecule, even though they are far too small to see directly.
Diffraction pattern
The pattern of spots or smears produced when X-rays scatter off atoms in a molecule and are captured on film or a detector. The positions and intensities of the spots encode information about the distances between atoms.
Photo 51
The famous X-ray diffraction image of B-form DNA taken by Rosalind Franklin and Raymond Gosling in May 1952. Its X-shaped pattern confirmed that DNA is a helix and provided key dimensional measurements.
A-form and B-form DNA
Two structural forms of DNA that depend on hydration. A-form is the "dry" form (below 75% humidity); B-form is the "wet" form found inside living cells. B-form produces the distinctive X-shaped diffraction pattern.
Dyad symmetry
The property of looking the same when flipped upside-down or front-to-back. DNA's dyad symmetry told Crick that the two strands must run in opposite directions.
Antiparallel
Running in opposite directions. In the double helix, one strand runs 5' to 3' and the other runs 3' to 5'. Think of it as two people standing side by side but facing opposite ways.
Triple-helix model
Watson and Crick's first (incorrect) model of DNA, consisting of three sugar-phosphate chains with bases on the outside. Franklin's data disproved it.
Tautomers
Alternate structural forms of the same molecule that differ in the position of a hydrogen atom. The "correct" (predominant) tautomers of guanine and thymine were essential for Watson to work out how the bases pair.
Hydrogen bond
A weak attractive force between a hydrogen atom bonded to an electronegative atom (like N or O) and another electronegative atom nearby. Hydrogen bonds hold the two strands of DNA together through base pairing: two bonds in an A-T pair, three in a G-C pair.
A beam of X-rays is directed at a sample. The X-rays bounce off atoms and scatter at angles determined by the distances between those atoms.
The scattered X-rays produce a diffraction pattern on photographic film. Mathematical calculations (Fourier transformations) convert these 2D patterns into a 3D model of the molecule.
For clear patterns, molecules must be arranged in a regular, repeating structure, which is why scientists crystallise molecules before analysis. Franklin used DNA fibres rather than crystals, which were regular enough to produce usable patterns.
William H. and William L. Bragg won the Nobel Prize (1915) for pioneering X-ray crystallography. Dorothy Hodgkin and Max Perutz later solved structures of complex organic molecules including insulin and haemoglobin.
Maurice Wilkins and Rosalind Franklin used X-ray crystallography at King's College London to study DNA's structure.
Franklin discovered that DNA exists in two forms depending on humidity:
A-form (dry, below 75% humidity): produces a scattered pattern with many distinct spots.
B-form (wet, above 75% humidity): produces the characteristic X-shaped diffraction pattern. This is the form found in living cells.
Franklin's Photo 51 (B-form, taken May 1952) was critical evidence. The X-shaped pattern confirmed a helical structure. Analysis of the pattern revealed: a radius of 1.0 nm, 0.34 nm between base pairs, and 3.4 nm (10 base pairs) per complete turn of the helix.
Franklin's images also revealed dyad symmetry, meaning the molecule looks the same flipped upside-down or front-to-back. Crick inferred from this that the two strands must run in opposite (antiparallel) directions.
Franklin's water-content measurements showed that phosphate groups were on the outside of the molecule, in contact with the aqueous environment.
Watson and Crick's first model proposed three sugar-phosphate chains in the centre, held together by magnesium ions, with bases projecting outward.
Franklin's critique demolished this model: DNA fibres contained far more water than the model allowed, there was no evidence for magnesium ions in DNA, and there was no explanation for how three phosphate chains could cluster at the centre.
Linus Pauling independently proposed his own triple-helix model with hydrogen bonds (rather than magnesium ions) holding the phosphate chains together. This model also failed on chemical grounds.
Watson and Crick tried again, this time placing two antiparallel sugar-phosphate backbones on the outside, with bases on the inside.
Using Chargaff's rules (A = T, G = C), Crick reasoned that A must pair with T and G with C.
A problem arose: Watson initially used the wrong tautomeric forms of guanine and thymine (taken from a 1950 textbook), which prevented the bases from forming proper hydrogen bonds.
Jerry Donohue, a visiting chemist, corrected the base structures. With the right tautomers, Watson saw that A-T and G-C pairs formed hydrogen bonds that matched the dimensions from Franklin's X-ray data.
The model was structurally stable, consistent with all known evidence, and elegantly explained how DNA could be replicated.
On the morning of 28 February 1953, Watson assembled the final model. Watson and Crick published their findings in Nature in April 1953, alongside corroborating papers by Wilkins (with Stokes and Wilson) and Franklin (with Gosling).
A-T pairs are held together by two hydrogen bonds.
G-C pairs are held together by three hydrogen bonds (Watson and Crick initially proposed two for both pairs; later evidence corrected the G-C count to three).
The A-T and G-C base pairs are the same width, which keeps the two backbones at a constant distance apart, producing the regular helical shape.
Students often think Watson and Crick did the X-ray crystallography themselves. They did not. The experimental X-ray work was done by Franklin, Gosling, and Wilkins at King's College. Watson and Crick built physical models at Cambridge.
Many students believe Watson and Crick went straight to the double helix. In reality, they first built an incorrect triple-helix model and only arrived at the double helix after incorporating new evidence and correcting earlier mistakes.
Students sometimes think the bases are on the outside of the DNA molecule. The bases are on the inside, paired together. The sugar-phosphate backbones form the exterior of the helix.
A common error is stating that A-T and G-C pairs both have the same number of hydrogen bonds. A-T pairs have two hydrogen bonds; G-C pairs have three.
⚠️ Know the key features of the double helix: two antiparallel sugar-phosphate backbones on the outside, complementary base pairs on the inside, held by hydrogen bonds.
⚠️ Be able to explain what Photo 51 showed and why the X-shaped diffraction pattern indicates a helix.
⚠️ Understand why Watson and Crick's triple-helix model failed and what evidence led them to revise it. This is a common "nature of science" exam question.
⚠️ Know the dimensions: 1.0 nm radius, 0.34 nm between base pairs, 3.4 nm per complete turn (10 base pairs per turn).
⚠️ Be able to explain why A pairs with T and G pairs with C (complementary shapes, correct hydrogen bonding with the predominant tautomeric forms).
True or False: Franklin used X-ray crystallography on DNA crystals. (False, she used DNA fibres.)
Fill in the blank: The B-form of DNA produces an ____-shaped diffraction pattern. (X-shaped.)
True or False: Watson and Crick's first model of DNA was a double helix. (False, it was a triple helix.)
Fill in the blank: An A-T base pair is held together by ____ hydrogen bonds, while a G-C pair has ____. (Two; three.)
True or False: In the double helix, both strands run in the same direction. (False, they are antiparallel.)
Q: Describe the process of X-ray crystallography and explain why it was useful for studying DNA.
A: X-ray crystallography involves directing a beam of X-rays at a sample. The X-rays scatter off atoms at angles determined by inter-atomic distances, producing a diffraction pattern on film. Mathematical analysis (Fourier transformations) converts these patterns into a 3D model. It was useful for DNA because the molecule is too small to see directly, but its repeating structure produces interpretable diffraction patterns.
Q: What key information did Photo 51 provide about the structure of DNA?
A: Photo 51's X-shaped diffraction pattern confirmed DNA is a helix. Analysis of the pattern revealed the molecule's dimensions: a radius of 1.0 nm, 0.34 nm between base pairs, and 3.4 nm (10 base pairs) per complete helical turn. It also indicated that the sugar-phosphate backbone is on the outside and the bases are on the inside.
Q: Why did Watson and Crick's triple-helix model fail? Give at least two reasons.
A: Franklin showed that DNA fibres contained far more water than the model allowed, the model required magnesium ions for which there was no evidence, and there was no explanation for how three phosphate chains could be held together at the centre.
Q: What role did Jerry Donohue play in solving the structure of DNA?
A: Donohue corrected the tautomeric forms of guanine and thymine that Watson was using. Chemistry textbooks of the time had the wrong forms. Once Watson used the correct (predominant) tautomers, the hydrogen bonding pattern between A-T and G-C base pairs fell into place.
Q: Explain what "antiparallel" means in the context of DNA and what evidence pointed to this arrangement.
A: Antiparallel means the two strands of DNA run in opposite directions (one 5' to 3', the other 3' to 5'). The evidence came from Franklin's observation that DNA has dyad symmetry, meaning it looks the same when flipped upside-down or front-to-back. Crick inferred that this symmetry required the strands to run in opposite orientations.
This material builds directly on Part 1 (the experiments proving DNA is the genetic material) and leads into Part 3 (the biological implications of DNA's structure for replication, information storage, and mutation). The concept of antiparallel strands is critical for understanding DNA replication, where the leading and lagging strands are synthesised differently. Hydrogen bonding between base pairs also applies to RNA secondary structure and to understanding how transcription and translation work.
X-ray crystallography, diffraction pattern, Photo 51, Rosalind Franklin, Maurice Wilkins, Watson and Crick, double helix, triple helix model, antiparallel strands, dyad symmetry, hydrogen bonds, base pairing, A-T pair, G-C pair, tautomers, A-form DNA, B-form DNA, Linus Pauling, Jerry Donohue, Fourier transformation, nucleotide dimensions, 0.34 nm, 3.4 nm, 10 base pairs per turn, sugar-phosphate backbone, Nature 1953, HHMI BioInteractive, the double helix film