Abstract
Metals have crucial roles in many physiological, pathological, toxicological, pharmaceutical, and diagnostic processes. Proper handling of metal-containing macromolecule samples for structural studies is not trivial, and failure to handle them properly is often a source of irreproducibility caused by issues such as pH changes, incorporation of unexpected metals, or oxidization/reduction of the metal. This protocol outlines the guidelines and best practices for characterizing metal-binding sites in protein structures and alerts experimenters to potential pitfalls during the preparation and handling of metal-containing protein samples for X-ray crystallography studies. The protocol features strategies for controlling the sample pH and the metal oxidation state, recording X-ray fluorescence (XRF) spectra, and collecting diffraction data sets above and below the corresponding metal absorption edges. This protocol should allow experimenters to gather sufficient evidence to unambiguously determine the identity and location of the metal of interest, as well as to accurately characterize the coordinating ligands in the metal binding environment within the protein. Meticulous handling of metal-containing macromolecule samples as described in this protocol should enhance experimental reproducibility in biomedical sciences, especially in X-ray macromolecular crystallography. For most samples, the protocol can be completed within a period of 7–190 d, most of which (2–180 d) is devoted to growing the crystal. The protocol should be readily understandable to structural biologists, particularly protein crystallographers with an intermediate level of experience.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Rötzschke, O., Lau, J.M., Hofstätter, M., Falk, K. & Strominger, J.L. A pH-sensitive histidine residue as control element for ligand release from HLA-DR molecules. Proc. Natl. Acad. Sci. USA 99, 16946–16950 (2002).
Holm, R.H., Kennepohl, P. & Solomon, E.I. Structural and functional aspects of metal sites in biology. Chem. Rev. 96, 2239–2314 (1996).
Pyle, A.M. Metal ions in the structure and function of RNA. J. Biol. Inorg. Chem. 7, 679–690 (2002).
Potter, J.D., Sheng, Z., Pan, B. & Zhao, J. A structural role for the Ca2+-Mg2+ sites on troponin regulation of muscle contraction. J. Biol. Chem. 270, 2557–2562 (1995).
Pace, N. & Weerapana, E. Zinc-binding cysteines: diverse functions and structural motifs. Biomolecules 4, 419–434 (2014).
McCall, K.A., Huang, C. & Fierke, C.A. Function and mechanism of zinc metalloenzymes. J. Nutr. 130, 1455–1458 (2000).
Solomon, E.I. et al. Copper active sites in biology. Chem. Rev. 114, 3659–3853 (2014).
MacPherson, I.S. & Murphy, M.E.P. Type-2 copper-containing enzymes. Cell. Mol. Life Sci. 64, 2887–2899 (2007).
Mccall, K.A., Huang, C.-C. & Fierke, C.A. Zinc and health: current status and future directions function and mechanism of zinc metalloenzymes 1. J. Nutr. 130, 1437–1446 (2000).
Lyons, T. & Eide, D. Transport and storage of metal ions in biology. in Biological Inorganic Chemistry: Structure and Reactivity (eds. Bertini, I. et al.) 57–78 (Univ. Sci. Books, 2007).
Farrell, N. Metal complexes as drugs and chemotherapeutic agents. in Comprehensive Coordination Chemistry II (eds. McCleverty, J.A. & Meyer. T.J.) 9, 809–840 (Elsevier Science, 2003).
Williams, C.J., Whitehouse, J.M.A. & Medical, B. Cis-platinum: a new anticancer agent. Br. Med. J. 1, 1689–1691 (1979).
Crack, J, Green, J., Thomson, A.J. & Le Brun, N.E. Techniques for the production, isolation, and analysis of iron–sulfur proteins. Methods Mol. Biol. 1122, 33–48 (2014).
Volbeda, A. X-ray crystallographic studies of metalloproteins. Methods Mol. Biol. 1122, 189–206 (2014).
Bowman, S.E.J., Bridwell-Rabb, J. & Drennan, C.L. Metalloprotein crystallography: more than a structure. Acc. Chem. Res. 49, 695–702 (2016).
Brink, A. & Helliwell, J.R. New leads for fragment-based design of rhenium/technetium radiopharmaceutical agents. IUCrJ 4, 283–290 (2017).
Helliwell, J.R. New developments in crystallography: exploring its technology, methods and scope in the molecular biosciences. Biosci. Rep. 37, BSR20170204 (2017).
Tanley, S.W.M., Schreurs, A.M.M., Kroon-Batenburg, L.M.J. & Helliwell, J.R. Re-refinement of 4g4a: room-temperature X-ray diffraction study of cisplatin and its binding to His15 of HEWL after 14 months chemical exposure in the presence of DMSO. Acta Crystallogr. F Struct. Biol. Commun. 72, 253–254 (2016).
Tanley, S.W.M., Schreurs, A.M.M., Kroon-Batenburg, L.M.J. & Helliwell, J.R. Re-refinement of 4xan: hen egg-white lysozyme with carboplatin in sodium bromide solution. Acta Crystallogr. F, Struct. Biol. Commun. 72, 251–252 (2016).
Russo Krauss, I. et al. Principles and methods used to grow and optimize crystals of protein-metallodrug adducts, to determine metal binding sites and to assign metal ligands. Metallomics 9, 1534–1547 (2017).
Frier, J.A. & Perutz, M.F. Structure of human foetal deoxyhaemoglobin. J. Mol. Biol. 112, 97–112 (1977).
Reeke, G.N.J., Becker, J.W. & Edelman, G.M. The covalent and three-dimensional structure of concanavalin A. IV. Atomic coordinates, hydrogen bonding, and quaternary structure. J. Biol. Chem. 250, 1525–1548 (1975).
Berman, H.M. et al. The protein data bank. Nucleic Acids Res. 28, 235–242 (2000).
Gatzeva-Topalova, P.Z., Warner, L.R., Pardi, A. & Carlos, M. Metalloproteomics: forward and reverse approaches in metalloprotein structural and functional characterization. Curr. Opin. Chem. Biol. 18, 1492–1501 (2011).
Hall, J.F. et al. Towards the high-throughput expression of metalloproteins from the Mycobacterium tuberculosis genome. J. Synchrotron Radiat. 12, 4–7 (2005).
Acton, A.Q. Metalloproteins: Advances in Research and Application: 2013 Edition (ScholaryEditions, 2013).
Nicolini, C. Molecular Manufacturing (Springer Science & Business Media, 2013).
Maret, W. Metalloproteomics, metalloproteomes, and the annotation of metalloproteins. Metallomics 2, 117–125 (2010).
Shi, W. et al. Characterization of metalloproteins by high-throughput X-ray absorption spectroscopy. Genome Res. 21, 898–907 (2011).
Macedo, S. et al. Can soaked-in scavengers protect metalloprotein active sites from reduction during data collection? J. Synchrotron Radiat. 16, 191–204 (2009).
Metalloproteins: Theory, Calculations, and Experiments (eds. Cho, A.E. & Goddard, W.A.) (CRC Press, 2015).
Domagalski, M. et al. The quality and validation of structures from structural genomics. Methods Mol. Biol. 1091, 297–314 (2013).
Zheng, H. et al. Validation of metal-binding sites in macromolecular structures with the CheckMyMetal web server. Nat. Protoc. 9, 156–170 (2014).
Riordan, J. & Valee, B. (eds.) Methods in Enzymology: Metallobiochemistry, Part C 226 (Academic Press, 1993).
Riordan, J. & Valee, B. (eds.) Methods in Enzymology: Metallobiochemistry, Part A 158 (Academic Press, 1988).
Riordan, J. & Valee, B. (eds.) Methods in Enzymology: Metallobiochemistry, Part D 227 (Academic Press, 1993).
Papageorgiou, A.C. & Mattsson, J. Protein structure validation and analysis with X-ray crystallography. Methods Mol. Biol. 1129, 397–421 (2014).
Shabalin, I., Dauter, Z., Jaskolski, M., Minor, W. & Wlodawer, A. Crystallography and chemistry should always go together: a cautionary tale of protein complexes with cisplatin and carboplatin. Acta Crystallogr. D Biol. Crystallogr. 71, 1965–1979 (2015).
Zimmerman, M.D., Proudfoot, M., Yakunin, A. & Minor, W. Structural insight into the mechanism of substrate specificity and catalytic activity of an HD-domain phosphohydrolase: the 5′-deoxyribonucleotidase YfbR from Escherichia coli. J. Mol. Biol. 378, 215–226 (2008).
Minor, W. et al. Crystal structure of soybean lipoxygenase L-1 at 1.4 A resolution. Biochemistry 35, 10687–10701 (1996).
Tomchick, D.R., Phan, P., Cymborowski, M., Minor, W. & Holman, T.R. Structural and functional characterization of second-coordination sphere mutants of soybean lipoxygenase-1. Biochemistry 40, 7509–7517 (2001).
Chruszcz, M., Wlodawer, A. & Minor, W. Determination of protein structures: a series of fortunate events. Biophys. J. 95, 1–9 (2008).
Saikatendu, K.S. et al. Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center. BMC Struct. Biol. 6, 27 (2006).
Niedzialkowska, E. et al. Molecular basis for phosphospecific recognition of histone H3 tails by Survivin paralogues at inner centromeres. Mol. Biol. Cell 23, 1457–1466 (2012).
Majorek, K.A., Kuhn, M.L., Chruszcz, M., Anderson, W.F. & Minor, W. Structural, functional, and inhibition studies of a Gcn5-related N-acetyltransferase (GNAT) superfamily protein PA4794: a new C-terminal lysine protein acetyltransferase from Pseudomonas aeruginosa. J. Biol. Chem. 288, 30223–30235 (2013).
Luo, H.-B. et al. Crystal structure and molecular modeling study of N-carbamoylsarcosine amidase Ta0454 from Thermoplasma acidophilum. J. Struct. Biol. 169, 304–311 (2010).
Murphy, T.A. et al. Crystal structure of Pseudomonas aeruginosa SPM-1 provides insights into variable zinc affinity of metallo-beta-lactamases. J. Mol. Biol. 357, 890–903 (2006).
Segraves, E.N. et al. Kinetic, spectroscopic, and structural investigations of the soybean lipoxygenase-1 first-coordination sphere mutant, Asn694Gly. Biochemistry 45, 10233–10242 (2006).
Zheng, H., Chruszcz, M., Lasota, P., Lebioda, L. & Minor, W. Data mining of metal ion environments present in protein structures. J. Inorg. Biochem. 102, 1765–76 (2008).
Zheng, H., Shabalin, I.G., Handing, K.B., Bujnicki, J.M. & Minor, W. Magnesium-binding architectures in RNA crystal structures: validation, binding preferences, classification and motif detection. Nucleic Acids Res. 43, 3789–3801 (2015).
Handing, K.B. et al. Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins. Chem. Sci. 7, 6635–6648 (2016).
Gabbiani, C. et al. Protein metalation by metal-based drugs: reactions of cytotoxic gold compounds with cytochrome c and lysozyme. J. Biol. Inorg. Chem. 17, 1293 (2012).
Merlino, A., Marzo, T. & Messori, L. Protein metalation by anticancer metallodrugs: a joint ESI MS and XRD investigative strategy. Chemistry 23, 6942–6947 (2017).
Allen, J.P. Biophysical Chemistry (Wiley-Blackwell, 2008).
Ostendorp, T., Diez, J., Heizmann, C.W. & Fritz, G. The crystal structures of human S100B in the zinc- and calcium-loaded state at three pH values reveal zinc ligand swapping. Biochim. Biophys. Acta 1813, 1083–1091 (2011).
Giroux, E.L. & Henkin, R.I. Competition for zinc among serum albumin and amino acids. Biochim. Biophys. Acta 273, 64–72 (1972).
Pantoliano, M.W., Valentine, J.S., Mammone, R.J. & Scholler, D.M. The pH dependence of metal ion binding to the native zinc site of bovine erythrocuprein (superoxide dismutase). J. Am. Chem. Soc. 104, 1717–1723 (1982).
McPherson, A. Current approaches to macromolecular crystallization. Eur. J. Biochem. 189, 1–23 (1990).
Newman, J., Sayle, R.A. & Fazio, V.J. A universal indicator dye pH assay for crystallization solutions and other high-throughput applications. Acta Crystallogr. D Biol. Crystallogr. 68, 1003–1009 (2012).
Newman, J., Fazio, V.J., Lawson, B. & Peat, T.S. The C6 web tool: a resource for the rational selection of crystallization conditions. Cryst. Growth Des. 10, 2785–2792 (2010).
Mikol, V., Rodeau, J.L. & Giegé, R. Changes of pH during biomacromolecule crystallization by vapor diffusion using ammonium sulfate as the precipitant. J. Appl. Crystallogr. 22, 155–161 (1989).
Hampton Research Corporation. PEG Stability (2012).
Orlov, Y.F., Maslov, E.I. & Belkina, E.I. Solubilities of metal hydroxides. Russ. J. Inorg. Chem. 58, 1458–1466 (2013).
Fischer, B.E., Häring, U.K., Tribolet, R. & Sigel, H. Metal ion/buffer interactions: stability of binary and ternary complexes containing 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) and adenosine 5-triphosphate (ATP). Eur. J. Biochem. 94, 523–530 (1979).
Belviso, B.D. et al. Oxaliplatin binding to human copper chaperone Atox1 and protein dimerization. Inorg. Chem. 55, 6563–6573 (2016).
David, G., Blondeau, K., Schiltz, M., Penel, S. & Lewit-Bentley, A. YodA from Escherichia coli is a metal-binding, lipocalin-like protein. J. Biol. Chem. 278, 43728–43735 (2003).
Cotelesage, J.J.H., Pushie, M.J., Grochulski, P., Pickering, I.J. & George, G.N. Metalloprotein active site structure determination: synergy between X-ray absorption spectroscopy and X-ray crystallography. J. Inorg. Biochem. 115, 127–137 (2012).
Ascone, I. & Strange, R. Biological X-ray absorption spectroscopy and metalloproteomics. J. Synchrotron Radiat. 16, 413–421 (2009).
Yukl, E.T. et al. Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis. Proc. Natl. Acad. Sci. USA 110, 4569–4573 (2013).
Garman, E.F. Radiation damage in macromolecular crystallography: what is it and why should we care? Acta Crystallogr. D Biol. Crystallogr. 66, 339–351 (2010).
Seitlich, T., Kühnel, K., Schulze-Briese, C., Shoeman, R.L. & Schlichting, I. Cryoradiolytic reduction of crystalline heme proteins: analysis by UV-Vis spectroscopy and X-ray crystallography. J. Synchrotron Radiat. 14, 11–23 (2007).
Frankaer, C.G., Mossin, S., Ståhl, K. & Harris, P. Towards accurate structural characterization of metal centres in protein crystals: the structures of Ni and Cu T6 bovine insulin derivatives. Acta Crystallogr. D Biol. Crystallogr. 70, 110–122 (2014).
Trofimov, A.A. et al. Structural study of the X-ray-induced enzymatic reaction of octahaem cytochrome C nitrite reductase. Acta Crystallogr. D 71, 1087–1094 (2015).
Brophy, M.B., Hayden, J.A. & Nolan, E.M. Calcium ion gradients modulate the zinc affinity and antibacterial activity of human calprotectin. J. Am. Chem. Soc. 134, 18089–18100 (2012).
Shirran, S., Garnaud, P., Daff, S., McMillan, D. & Barran, P. The formation of a complex between calmodulin and neuronal nitric oxide synthase is determined by ESI-MS. J. R. Soc. Interface 2, 465–476 (2005).
Kaltashov, I.A., Zhang, M., Eyles, S.J. & Abzalimov, R.R. Investigation of structure, dynamics and function of metalloproteins with electrospray ionization mass spectrometry. Anal. Bioanal. Chem. 386, 472–481 (2006).
Czernusxewicx, R.S. in Spectroscopic Methods and Analyses: NMR, Mass Spectrometry, and Metalloprotein Techniques (eds. Jones, C. et al.) 345–374 (Humana Press, 1993).
Vincent, K. Triggered infrared spectroscopy for investigating metalloprotein chemistry. Philos. Trans. A Math. Phys. Eng. Sci. 368, 3713–3731 (2010).
Ronda, L., Bruno, S., Bettati, S., Storici, P. & Mozzarelli, A. From protein structure to function via single crystal optical spectroscopy. Front. Mol. Biosci. 2, 12 (2015).
Stoner-Ma, D. et al. Single-crystal Raman spectroscopy and X-ray crystallography at beamline X26-C of the NSLS. J. Synchrotron Radiat. 18, 37–40 (2011).
Merlino, A. et al. Crystallization, preliminary X-ray diffraction studies and Raman microscopy of the major haemoglobin from the sub-Antarctic fish Eleginops maclovinus in the carbomonoxy form. Acta Crystallogr. F Struct. Biol. Cryst. Commun. 66, 1536–1540 (2010).
Vergara, A., Merlino, A., Pizzo, E., D'Alessio, G. & Mazzarella, L. A novel method for detection of selenomethionine incorporation in protein crystals via Raman microscopy. Acta Crystallogr. D 64, 167–171 (2008).
Hall, J.P. et al. Monitoring one-electron photo-oxidation of guanine in DNA crystals using ultrafast infrared spectroscopy. Nat. Chem. 7, 961–967 (2015).
Knape, M.J. et al. Divalent metal ions Mg2+ and Ca2+ have distinct effects on protein kinase A activity and regulation. ACS Chem. Biol. 10, 2303–2315 (2015).
Lu, C.H., Lin, Y.F., Lin, J.J. & Yu, C.S. Prediction of metal ion-binding sites in proteins using the fragment transformation method. PLoS One 7, 1–12 (2012).
Ebert, J.J.C. & Altman, R.R.B. Robust recognition of zinc binding sites in proteins. Protein Sci. 17, 54–65 (2008).
Zhao, W. et al. Structure-based de novo prediction of zinc-binding sites in proteins of unknown function. Bioinformatics 27, 1262–1268 (2011).
Sodhi, J.S. et al. Predicting metal-binding site residues in low-resolution structural models. J. Mol. Biol. 342, 307–320 (2004).
Passerini, A., Lippi, M. & Frasconi, P. MetalDetector v2.0: predicting the geometry of metal binding sites from protein sequence. Nucleic Acids Res. 39, 288–292 (2011).
Ferrè, F. & Clote, P. DiANNA 1.1: an extension of the DiANNA web server for ternary cysteine classification. Nucleic Acids Res. 34, W182–W185 (2006).
Brylinski, M. & Skolnick, J. FINDSITE-metal: integrating evolutionary information and machine learning for structure-based metal binding site prediction at the proteome level. Proteins 79, 735–751 (2011).
Alpi, E. et al. Analysis of the tryptic search space in UniProt databases. Proteomics 15, 48–57 (2015).
Chitale, M., Hawkins, T., Park, C. & Kihara, D. ESG: extended similarity group method for automated protein function prediction. Bioinformatics 25, 1739–1745 (2009).
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
Leuthner, B. et al. Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism. Mol. Microbiol. 28, 615–628 (1998).
Echavarri, C., Arragain, S. & Rubio, L.M. Purification of O2-sensitive metalloproteins. Methods Mol. Biol. 1122, 5–18 (2014).
Pestov, N.B. & Rydstro, J. Purification of recombinant membrane proteins tagged with calmodulin-binding domains by affinity chromatography on calmodulin-agarose: example of nicotinamide nucleotide transhydrogenase. Nat. Protoc. 2, 198–202 (2007).
Majorek, K.A., Kuhn, M.L., Chruszcz, M., Anderson, W.F. & Minor, W. Double trouble-buffer selection and His-tag presence may be responsible for nonreproducibility of biomedical experiments. Protein Sci. 23, 1359–1368 (2014).
Van Dam, M.E., Wuenschell, G.E. & Arnold, F.H. Metal affinity precipitation of proteins. Biotechnol. Appl. Biochem. 11, 492–502 (1989).
Ju, T. et al. One protein, two enzymes revisited: a structural entropy switch interconverts the two isoforms of acireductone dioxygenase. J. Mol. Biol. 4, 823–834 (2011).
Smith, R.M., Martell, A.E. & Motekaitis, R.J. NIST critically selected stability constants of metal complexes database (Standard Reference Data Program, National Institute of Standards and Technology, U.S. Dept. of Commerce 2004).
Bijelic, A., Theiner, S., Keppler, B.K. & Rompel, A. X-ray structure analysis of indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) bound to human serum albumin reveals two ruthenium binding sites and provides insights into the drug binding mechanism. J. Med. Chem. 59, 5894–5903 (2016).
Yu, Q., Kandegedara, A., Xu, Y. & Rorabacher, D.B. Avoiding interferences from Good's buffers: a contiguous series of noncomplexing tertiary amine buffers covering the entire range of pH 3-11. Anal. Biochem. 253, 50–56 (1997).
Nakon, R. & Krishnamoorthy, C.R. Free-metal ion depletion by 'Good's' buffers. Science 221, 749–750 (1983).
Sokołowska, M. & Bal, W. Cu(II) complexation by 'non-coordinating' N-2- hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES buffer). J. Inorg. Biochem. 99, 1653–1660 (2005).
Ferreira, C.M.H., Pinto, I.S.S., Soares, E.V. & Soares, H.M.V.M. (Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ions: a review. RSC Adv. 5, 30989–31003 (2015).
Lundblad, R.L. Chemical Reagents for Protein Modification (CRC Press, 2014).
Satyanarayana, U. Biochemistry (Elsevier, 2014).
Phillips, C.M., Schreiter, E.R., Stultz, C.M. & Drennan, C.L. Structural basis of low-affinity nickel binding to the nickel-responsive transcription factor NikR from Escherichia coli. Biochemistry 49, 7830–7838 (2010).
Champloy, F., Gruber, K., Jogl, G. & Kratky, C. XAS spectroscopy reveals X-ray-induced photoreduction of free and protein-bound B12 cofactors. J. Synchrotron Radiat. 7, 267–273 (2000).
Huang, Q. & Szebenyi, D.M.E. Improving diffraction resolution using a new dehydration method. Acta Crystallogr. F Struct. Biol. Commun. 72, 152–159 (2016).
Newman, J. A review of techniques for maximizing diffraction from a protein crystal in stilla. Acta Crystallogr. D 62, 27–31 (2006).
Kim, C.U., Wierman, J.L., Gillilan, R., Lima, E. & Gruner, S.M. A high-pressure cryocooling method for protein crystals and biological samples with reduced background X-ray scatter. J. Appl. Crystallogr. 46, 234–241 (2013).
Vernède, X. & Fontecilla-Camps, J.C. A method to stabilize reduced and/or gas-treated protein crystals by flash-cooling under a controlled atmosphere. J. Appl. Crystallogr. 32, 505–509 (1999).
Bearden, J.A. X-ray wavelengths. Rev. Mod. Phys. 39, 78–124 (1967).
Nakanishi, T. et al. Lower limits of detection of synchrotron radiation high-energy X-ray fluorescence spectrometry and its possibility for the forensic application for discrimination of glass fragments. Forensic Sci. Int. 175, 227–234 (2008).
Awaji, N. et al. Detection limits of trace elements for wavelength dispersive total X-ray fluorescence under high flux synchrotron radiation. Jpn. J. Appl. Phys. 43, 1644–1648 (2004).
Sutton, S.R., Rivers, M.L. & Smith, J.V. Synchrotron X-ray fluorescence: diffraction interference. Anal. Chem. 58, 2167–2171 (1986).
Brock, C.P. et al. (eds.) International Tables for Crystallography. Vol. C(Wiley, 1999).
Srivastava, U.C. & Nigam, H.L. X-ray absorption edge spectrometry (XAES) as applied to coordination chemistry. Coord. Chem. Rev. 9, 275–310 (1973).
Sathyanarayana, D.N. Electronic Absorption Spectroscopy and Related Techniques (Universities Press, 2001).
Cotelesage, J.J.H., Grochulski, P., Pickering, I.J., George, G.N. & Fodje, M.N. X-ray absorption spectroscopy at a protein crystallography facility: the Canadian Light Source beamline 08B1-1. J. Synchrotron Radiat. 19, 887–891 (2012).
Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).
Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).
Zheng, H. et al. CheckMyMetal: macromolecular metal binding validation tool. Acta Crystallogr. D 73, 223–233 (2017).
Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).
Gore, S., Velankar, S. & Kleywegt, G.J. Implementing an X-ray validation pipeline for the Protein Data Bank. Acta Crystallogr. D Biol. Crystallogr. 68, 478–483 (2012).
Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. HKL-3000: the integration of data reduction and structure solution: from diffraction images to an initial model in minutes. Acta Crystallogr. D Biol. Crystallogr. 62, 859–866 (2006).
Stepanov, S. et al. JBluIce: EPICS control system for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 67, 176–188 (2011).
Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).
Adams, P.D. et al. PHENIX: a comprehensive python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Bruvold, W.H. A meta-analysis of the California school-based risk reduction program. J. Drug Educ. 20, 139–152 (1990).
Kimple, M.E., Brill, A.L. & Pasker, R.L. Overview of affinity tags for protein purification. Curr. Protoc. Protein Sci. 36 9.9 (2013).
Bonner, P.L.R. Protein Purification (Taylor & Francis, 2007).
Scopes, R.K. Protein Purification: Principles and Practice (Springer, 1993).
Ammann, A.A. Inductively coupled plasma mass spectrometry (ICP MS): a versatile tool. J. Mass Spectrom. 42, 419–427 (2007).
McPherson, A. Crystallization of Biological Macromolecules (Cold Spring Harbor Laboratory Press, 1999).
Pflugrath, J.W. Macromolecular cryocrystallography: methods for cooling and mounting protein crystals at cryogenic temperatures. Methods 34, 415–423 (2004).
Tereshko, V. et al. Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments. Nucleic Acids Res. 29, 1208–15 (2001).
Niedzialkowska, E. et al. Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization. Protein Expr. Purif. 134, 47–62 (2017).
Lion, T. et al. The translocation t(1;22)(p13;q13) is a nonrandom marker specifically associated with acute megakaryocytic leukemia in young children. Blood 79, 3325–3330 (1992).
Blindauer, C.A. et al. Structure, properties, and engineering of the major zinc binding site on human albumin. J. Biol. Chem. 284, 23116–23124 (2009).
Newman, J. Expanding screening space through the use of alternative reservoirs in vapor-diffusion experiments. Acta Crystallogr. D Biol. Crystallogr. 61, 490–493 (2005).
Till, M. et al. Improving the success rate of protein crystallization by random microseed matrix screening. J. Vis. Exp. http://doi.org/10.3791/50548 (2013).
Benvenuti, M. & Mangani, S. Crystallization of soluble proteins in vapor diffusion for X-ray crystallography. Nat. Protoc. 2, 1633–1651 (2007).
Malawski, G.A. et al. Identifying protein construct variants with increased crystallization propensity: a case study. Protein Sci. 15, 2718–2728 (2006).
Vedadi, M. et al. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc. Natl. Acad. Sci. USA 103, 15835–15840 (2006).
McPherson, A. & Cudney, B. Optimization of crystallization conditions for biological macromolecules. Acta Crystallogr. F Struct. Biol. Commun. 70, 1445–1467 (2014).
Dong, A., Xu, X. & Edwards, A. In situ proteolysis for protein crystallization and structure determination. Nat. Methods 4, 1019–1021 (2007).
Holdgate, G. Isothermal titration calorimetry and differential scanning calorimetry. Methods Mol. Biol. 572, 101–133 (2009).
Acknowledgements
This work was supported by federal funds awarded to W.M. from the National Institute of General Medical Sciences under grant numbers GM117325 and GM117080, and NIH BD2K grant HG008424, as well as from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and the Department of Health and Human Services under contract nos. HHSN272201200026C and HHSN272201700060C. E.N. was supported by the Foundation for Polish Science (FNP) and received funding from the Marian Smoluchowski Krakow Research Consortium—a Leading National Research Centre KNOW supported by the Polish Ministry of Science and Higher Education. We thank J. Lipowska for providing the fluorescence and diffraction data for dihydroorotase from Y. pestis CO92. We thank M.P. Czub for providing Thermofluor shift data for STM1931 protein from S. Typhimurium. We thank R. Alkire (Structural Biology Center at Argonne National Laboratory) for providing the fluorescence spectrum data for the zinc foil. We thank D.R. Cooper and M. Cymborowski for help with XRF data collection and interpretation. We thank M. Grabowski, W.-S. Tzou, and B. Venkataramany for valuable discussions and critical reading of the manuscript.
Author information
Authors and Affiliations
Contributions
K.B.H. performed essential experiments to identify the pH-dependent conformational changes of residues coordinating the transition metal in albumin; E.N. provided important data and experience in ICP–OES and TSA experiments; K.B.H., I.G.S., E.N., M.L.K., and H.Z. provided critical experience in metal-binding-protein production and purification; K.B.H., I.G.S., E.N., and W.M. provided critical experience in metal-binding-protein crystallization and data collection; H.Z. and I.G.S. provided extensive experience in characterization of metal-binding sites in protein structures; H.Z. and I.G.S. laid out the structural framework of the manuscript; M.L.K. provided constructive comments and extensively edited the manuscript; K.B.H., E.N., I.G.S., M.L.K., H.Z., and W.M. wrote and approved the manuscript; and H.Z. and W.M. supervised the project.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Influence of ZnCl2 concentration on pH of 50 (gray) and 100 mM (orange) Tris-HCl buffer solutions.
The green line represents the initial value of the pH of Tris buffers.
Supplementary Figure 2 Fluorescence spectra and fluorescence scans for ESA complexed with four different metals.
Data for ESA complexed with copper (A), gold (B), mercury (C), and platinum (D) are shown. The figure represents screenshots generated by the data collection program JBluIce-EPICS as implemented on APS GM/CA-CAT beamlines. For each panel, the top screenshot shows the fluorescence emission spectrum collected with the excitation energy on or slightly above the theoretical value of the metal absorption edge (+10-20 eV). This spectrum displays the characteristic emission peak for the metal of interest (bracketed by the red boundaries) as well as the incident beam peak. Note that there are no significant peaks for other metals in these spectra. The middle screenshot shows the fluorescence emission spectrum collected with the excitation energy at 30-50 eV below the metal absorption edge; the characteristic peak for the metal of interest is absent on this spectrum. The bottom screenshot shows the fluorescence absorption scan collected with the excitation energy in the range ±30 eV of the tabulated metal absorption edge; note that the emitted fluorescence is measured, which is proportional to the absorbed energy. The energy of the absorption edge, approximated as the inflection f” point (indicated by the orange vertical line and listed in the table below each graph as “infl”), is close to the table values for each metal. Note that the width of the absorption edges (the energy difference between the absorbance inflection point and its peak; these values coincide with f” inflection point and its peak) for both Cu K edge and Hg L-III edge is much wider than those for Au L-III and Pt L-III. In the case of Hg L-III edge, the range of the excitation energy could be increased since the typical range of ±30 eV does not fully cover the absorption edge. The optimal energy for collecting X-ray diffraction data above the absorption edge is the maximum of f” (i.e. the maximum of absorption and fluorescence), which is located at the top of the fluorescence scan and indicated by the green vertical line. The optimal energy for collecting X-ray diffraction data below the absorption edge is the highest energy below the absorption edge that gives only background fluorescence signal (virtually flat area of f”).
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1 and 2, and Supplementary Tables 1–4 (PDF 1048 kb)
Rights and permissions
About this article
Cite this article
Handing, K., Niedzialkowska, E., Shabalin, I. et al. Characterizing metal-binding sites in proteins with X-ray crystallography. Nat Protoc 13, 1062–1090 (2018). https://doi.org/10.1038/nprot.2018.018
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2018.018
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.