An electron microscope can observe nanometre-size structures, but intense electron beams can damage samples and blur images. Klug overcame these issues by using the regular two-dimensional crystals formed by viruses or nucleic-acid–protein complexes and by extracting structural information mathematically; he named the approach “crystallographic electron microscopy.” Applying the Fourier transform you learn in high school, he calculated near-atomic density maps from diffraction patterns recorded on micrographs—a revolutionary step. This technique yielded the first detailed 3-D structures of biological giants such as tobacco mosaic virus and the nucleosome. With accurate shapes in hand, models of gene reading and viral infection became much more precise. The technology also opened the door to structure-based drug design, speeding the development of antiviral and anticancer agents. Thus Klug’s work benefits society by advancing infectious-disease control and the biotechnology industry.

Electron crystallography, as pioneered by Klug, applies electron microscopy to two-dimensional crystals or helical assemblies and reconstructs 3-D density from diffraction data. He introduced the Fourier-Bessel treatment of helical specimens and achieved high-resolution maps of tobacco mosaic virus. The approach could handle huge complexes that resisted X-ray crystallography, a decisive advantage at the time. Klug also developed algorithms that combine images taken at different tilt angles, enabling quasi-atomic reconstructions of nucleic-acid–protein assemblies. The elucidation of the nucleosome furnished a molecular basis for chromatin folding and transcriptional regulation, igniting the field of epigenetics. His concepts evolved into cryo-EM and single-particle analysis, which now dominate molecular structural biology. Because electrons scatter more strongly than X rays, thin samples yield intense signals, but multiple scattering and radiation damage had to be resolved; Klug handled these issues with image processing and physical modelling. With increased computing power the methods became automated, and 3–4 Å resolution virus-capsid structures became routine. Structural knowledge propelled functional analyses and rational design across chemistry, life science and medicine. In university courses, the technique is presented as a synthesis of electron scattering physics, Fourier space mathematics and biomolecular symmetry operations. Klug’s cautious attitude toward sample preparation and data credibility also influenced safety standards and good-practice guidelines. Hence his achievements underpin modern bionanoscience through technological innovation and interdisciplinary integration.

During the early cryo-EM era, Klug established a reconstruction pipeline that combined CTF correction with Fourier synthesis. His helical reconstruction method automated phase assignment of Bessel coefficients and reached 3.6 Å for TMV and ~7 Å for stretched nucleosomes. Addressing the missing-cone constraint, he proposed Fourier-space filling with random-tilt sets and quantified resolution via FSC—decades ahead of its widespread use. His group crystallised RNP particles into two-dimensional arrays, extracted accurate structure factors from electron diffraction, and performed parallel real- and reciprocal-space refinement. To mitigate radiation damage he collected low-dose images (<10 e⁻/Ų) and enhanced SNR through phase-flipped averaging and adjacent-frame difference methods. The outcome shaped EM data-base construction at EMBL-EBI and fostered FORTRAN-based software such as MRC and Bsoft. Cross-validation with electron tomography located heterodimer subunits and revealed functional asymmetry. On the theoretical side he treated dynamical scattering terms for multiple scatterers via Born-approximation corrections and fitted anisotropic scattering factors. These contributions laid the groundwork for maximum-likelihood alignment and 3-D classification that drive modern cryo-EM single-particle analysis. Klug thereby provided a roadmap that fuses X-ray crystallography, electron tomography and cryo-EM SPA, accelerating the so-called resolution revolution. His electron crystallography formed a unified school covering sample preparation to algorithm design and remains the lingua franca of high-resolution EM research.