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Macromolecular crystallography

Nature Reviews Methods Primers volume 5, Article number: 64 (2025) Cite this article

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An Author Correction to this article was published on 31 October 2025

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Abstract

Crystallography provides structural evidence of macromolecules in atomic detail. However, the atomic structure is not the direct outcome of the experiment. Diffraction data need to be processed and the phase problem must be solved to visualize the map from which an atomic model of the macromolecule is interpreted and iteratively improved. Despite this complex process from sample to scientific answer, crystallography is widely accessible in biochemical and biological research. Easy access to the experimental set-ups, free software for academic use and complimentary analytical computing, supported by automation and expert assistance, makes crystallography available to non-crystallographers. This Primer offers a practical and rational introduction to macromolecular crystallography, whether to engage directly or to critically assess results, with a focus on understanding the diffraction data, solving the phase problem, building and refining the atomic model, and interpreting the resulting atomic structure. We provide an overview of what crystallography can achieve, the key decisions and trade-offs involved, and how to evaluate outcomes effectively.

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Fig. 1: Overview of the macromolecular crystallography experiment and key fundamental concepts.
Fig. 2: X-ray diffraction patterns with 1° crystal rotation.
Fig. 3: Properties of the Fourier transform and implications for resolution and errors in crystallography.
Fig. 4: Crystallographic structure determination of oligopeptide-binding proteins.
Fig. 5: The importance in visual inspection of crystal structures.

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Acknowledgements

The authors thank all the lecturers that have contributed to the Madrid Crystallography School, including M. Martínez-Ripoll, F. X. Gomis-Rüth, J. M. García-Ruiz, R. Kahn, J. Navaza, C. Giacovazzo, P. Emsley, J. M. Mancheño, P. Adams, T. Grüne, I. Muñoz, P. Bernadó, R. Nicholls, R. Marabini, J. M. Carazo, J. Martín-Garcia, R. Fernández-Leiro, R. Boer, A. J. McCoy, B. Herguedas, T. T. Terwilliger, M. Fando, F. Sánchez-Rodríguez, R. Keegan and L. Catapano. They also thank all students for their active participation in discussions and contribution of interesting crystallographic challenges. This work was supported by (Ministry of Science and Innovation/Spanish State Research Agency/European Regional Development Fund/European Union) grants PID2021-128751NB-I00 to I.U., PID2023-153118OB-I00 to J.A.H., PID2023-153108OB-I00 to A.A. and PID2021-129038NB-I00 to M.S.; grant 2021-SGR-00425 (AGAUR) to I.U. and M.S.; grant Horizon Europe ID 101094131 and 101046133 to J.A.M.; The National Institutes of Health (grants R01GM071939, P01GM063210 and R24GM141254), as well as support from the Phenix Industrial Consortium and the US Department of Energy under contract no. DE-AC02-05CH11231 to P.V.A. The German Federal Ministry of Education and Research (grant no. 05K19WWA), Deutsche Forschungsgemeinschaft (project TH2135/2-1) to A.T. The Collaborative Computational Project Number 4 in Protein Crystallography (CCP4) and Biotechnology and Biological Research Council (BBSRC UK) grants BB/Y009991/1, BB/V015591/1 and BB/S007040/1 to E.K.

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Authors and Affiliations

  1. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Pavel V. Afonine

  2. Instituto de Química Física Blas Cabrera, Consejo Superior de Investigaciones Científicas, Madrid, Spain

    Armando Albert & Juan A. Hermoso

  3. Fachbereich Biologie, Universität Konstanz, Konstanz, Germany

    Kay Diederichs

  4. Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK

    Eugene Krissinel

  5. European Molecular Biology Laboratory, Grenoble, France

    José Antonio Márquez

  6. Australian Synchrotron, Melbourne, Victoria, Australia

    Santosh Panjikar

  7. Monash University, Melbourne, Victoria, Australia

    Santosh Panjikar

  8. Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona, Spain

    Maria Solà & Isabel Usón

  9. Institute for Nanostructure and Solid State Physics, Universität Hamburg, Hamburg, Germany

    Andrea Thorn

  10. Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain

    Isabel Usón

Authors
  1. Pavel V. Afonine
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  2. Armando Albert
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  3. Kay Diederichs
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  4. Juan A. Hermoso
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  5. Eugene Krissinel
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  6. José Antonio Márquez
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  7. Santosh Panjikar
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  8. Maria Solà
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  9. Andrea Thorn
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  10. Isabel Usón
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Contributions

Introduction (A.A., J.A.H., K.D. and I.U.); Experimentation (M.S., J.A.M., S.P., K.D. and I.U.); Results (P.V.A., K.D. and I.U.); Applications (J.A.H., E.K., K.D. and I.U.); Reproducibility and data deposition (A.T., K.D. and I.U.); Limitations and optimizations (K.D. and I.U.); Outlook (K.D. and I.U.); overview of the Primer (K.D. and I.U.).

Corresponding author

Correspondence to Isabel Usón.

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The authors declare no competing interests.

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Nature Reviews Methods Primers thanks Elspeth Garman, José Gavira and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

CCP4 Cloud: https://cloud.ccp4.ac.uk

Crystallographic wiki: https://wiki.uni-konstanz.de/ccp4/index.php/Main_Page

CSIC Crystallography: https://www.xtal.iqfr.csic.es/Cristalografia/index-en.html

Fourier transform animations: http://chango.ibmb.csic.es/colibri

International Union of Crystallography (IUCr) dictionary: https://dictionary.iucr.org

MolProbity analysis: http://molprobity.biochem.duke.edu/

Protein Data Bank: https://www.wwpdb.org

Radiation dose calculation: https://raddo.se/

SBGRID: https://SBGrid.org

The PDBe Knowledge base: https://www.ebi.ac.uk/pdbe/pdbe-kb/

Zenodo: https://zenodo.org

Supplementary information

Glossary

Arcimboldo methods

Phasing approaches that use small model fragments (such as α-helices) combined with density modification and fragment expansion to solve structures.

Asymmetric unit

The simply connected smallest closed part of space from which, by application of all symmetry operations of the space group, the whole space is filled.

Co-crystallization

The process of crystallizing two or more molecules together, often a macromolecule with a ligand or inhibitor to view their interaction; with cryoprotectant to minimize stress on fragile crystals or with heavier elements to modify diffraction.

Conjugate gradient minimization

An optimization algorithm used in structure refinement to minimize the difference between observed and calculated structure factors by adjusting model parameters.

Cryogenic cooling device

An apparatus (usually using liquid nitrogen) to rapidly cryo-cool crystals and/or maintain them at the low temperature required to reduce radiation damage during X-ray exposure.

Crystallization drop

A (sub)microlitre droplet containing protein, buffer and precipitant that is used to grow protein crystals.

Diffraction limit

The maximum resolution (smallest detail) that can be resolved in a crystal structure, determined by the quality of the crystal and data.

Direct methods

A set of computational techniques exploiting statistical relationships among structure factors, used to solve the phase problem ab initio and in experimental phasing.

Electron density distribution

A 3D map of the asymmetric unit showing electron per cubic ångström levels, used to model atomic positions and establish structural features.

Friedel opposites

Pairs of reflections related by inversion in reciprocal space (for example, (h,k,l) and (–h,–k,–l); their intensities are equal in the absence of anomalous scattering.

Goniometer

A precision device that holds the crystal and allows its controlled rotation during X-ray diffraction data collection.

Laue groups

Symmetry classifications based on the diffraction pattern, considering the point group of the crystal without translational symmetry.

Model bias

The electron density map is calculated with experimental amplitudes but phases derived from the current model. Hence, the model influences the map and errors can mask real structural features.

Monochromatic

Radiation of a single wavelength, typically used for high-resolution diffraction experiments.

Mosaicity

A measure of the spread of crystal plane orientations, given by the rotation angle over which the signal corresponding to a Bragg reflection is distributed.

Non-crystallographic symmetry

Occurs when copies of a molecule in the asymmetric unit are related by a rotation or translation that is not a symmetry operation of the crystal space group. Translational non-crystallographic symmetry causes aberrant diffraction and complicates structure solution.

Phase problem

The inability to directly measure the phase component of diffracted X-rays (neutrons or electrons), which is essential for reconstructing electron density maps. Phasing means providing approximate values for enough phases to be able to reconstruct an initial model of the structure in the crystal.

Polychromatic

Radiation composed of multiple wavelengths; often used in Laue diffraction experiments.

Real space

The physical, 3D coordinate system in which atoms and electron densities are located within a crystal structure.

Reciprocal space

An abstract space used in crystallography where diffraction data are represented; each point in a reciprocal space lattice corresponds to a set of planes in real space.

Structure factors

Mathematical complex quantities describing both the amplitude and phase of diffracted X-rays, calculated as a vector sum of atomic contributions within the unit cell.

Systematic grid searches

Automated exploration of crystallization conditions by systematically varying parameters such as pH, temperature and precipitant concentration. Also, automatic exploration of parameters in computational procedures like refinement and molecular replacement.

Voxel

A 3D pixel representing a small volume element in an electron density map.

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Afonine, P.V., Albert, A., Diederichs, K. et al. Macromolecular crystallography. Nat Rev Methods Primers 5, 64 (2025). https://doi.org/10.1038/s43586-025-00433-8

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