Those would have pretty significant signals in the difference density if fully occupied, but partial contamination may be possible. I suspect possibly Co or Zn (and may get shot down by the bioinorganic chemists), but without experimental confirmation, it's difficult to say. I always recommend an excitation scan after the data collection, it costs nothing but time, and as an atomic technique does not need the crystal to diffract anymore, or even a dedicated study such as Fruncillo et al. 2019 (https://pubs.acs.org/doi/10.1021/acs.analchem.9b03319). We’ve made good use of particle-induced X-ray emission at the Surrey Ion Beam Center to study individual metals in metalloproteins (https://pubs.acs.org/doi/pdf/10.1021/jacs.9b09186), and have some more news coming out on this shortly with X-ray fluorescence spectroscopy. Errors in metals are not uncommon, but they are rarer in well-defined cofactors. It’s better to confirm the metal at the beamline before wondering what the density is after, but I think in this case you can easily be forgiven 😊 There are many specialized metalloprotein laboratories that characterize their metals exquisitely; unfortunately, there are many more metalloproteins than those specialized laboratories 😊
Good luck! Eddie Edward Snell Ph.D. Professor, Materials Design and Innovation | University at Buffalo, SUNY Director | NSF BioXFEL Science and Technology Center Fellow of the American Crystallographic Society – The Structural Science Society p: +1 716 881 7573 (new) | c: +1 716 989 9128 | f: +1 716 898 8660 e: esnell@ buffalo.edu<mailto:[email protected]> The University at Buffalo Hauptman-Woodward Institute 700 Ellicott Street | Buffalo, NY 14203-1102 Website: https://snelllab.website/ [hwi-logo-primary-horizontal] From: CCP4 bulletin board <[email protected]> On Behalf Of Oganesyan, Vaheh Sent: Friday, September 5, 2025 11:23 AM To: [email protected] Subject: Re: [ccp4bb] Green density over Fe in heme One more less probable cause: could the haem be contaminated with metals heavier than Fe? I’ve asked that question to Gemini. Below is the response: Yes, a haem preparation can be contaminated with me This sender is trusted. sophospsmartbannerend One more less probable cause: could the haem be contaminated with metals heavier than Fe? I’ve asked that question to Gemini. Below is the response: Yes, a haem preparation can be contaminated with metals heavier than iron, including toxic heavy metals like lead (Pb), mercury (Hg), cadmium (Cd), and arsenic (As). This is a serious concern because these metals can interfere with normal heme synthesis and cause a range of health issues. Mechanisms of contamination Several pathways can lead to haem preparation contamination with heavy metals: * Environmental pollution and bioaccumulation: Heavy metals from industrial activity, mining, and waste disposal are persistent in the environment. These metals bioaccumulate in the food chain and can end up in the biological tissues from which haem is extracted. * Competition with iron in the body: The heavy metals that contaminate biological samples can interfere with the final step of heme biosynthesis, where iron is normally inserted into the protoporphyrin ring. Lead, for example, is known to inhibit the enzyme ferrochelatase, disrupting the process. * Experimental reagents: Contaminated reagents or laboratory equipment used during the haem preparation and purification process can introduce heavy metals. Even analytical methods, such as those that involve acid digestion, must be carefully controlled to prevent external contamination during analysis. Vaheh From: CCP4 bulletin board <[email protected]<mailto:[email protected]>> On Behalf Of Catherine Back Sent: Friday, September 5, 2025 8:51 AM To: [email protected]<mailto:[email protected]> Subject: Re: [ccp4bb] Green density over Fe in heme Hi All, Many thanks for all your suggestions. I've had some direct replies as well as all the ones from the BB. * The occupancies were definitely 1.0 * I'll refine anisotropically from now on. * The Bfactors for two of the Fe-atoms were abnormally high, which contributed to the density. I've altered them and they've gone back down to similar levels to the surrounding atoms. * The collection wavelength was 0.976 Å. * I was using Phenix Refine, though I tried Refmac first. There is still a little positive density in the area of the Fe, but the structure is well refined, so possibly this is just amplifying any tiny errors? Cheers, Catherine Dr Catherine R. Back (she/her) Senior Post-doctoral Research Associate School of Biochemistry University of Bristol UK ________________________________ From: CCP4 bulletin board <[email protected]<mailto:[email protected]>> on behalf of Jon Cooper <[email protected]<mailto:[email protected]>> Sent: 05 September 2025 13:17 To: [email protected]<mailto:[email protected]> <[email protected]<mailto:[email protected]>> Subject: Re: [ccp4bb] Green density over Fe in heme Treating the iron (Fe) as fluorine (F) is roughly equivalent having the iron occupancy at (9/26) or 0.34 approx ;-0 Best wishes, Jon Cooper. [email protected]<mailto:[email protected]> Sent from Proton Mail Android -------- Original Message -------- On 05/09/2025 13:03, Garib Murshudov <[email protected]<mailto:[email protected]>> wrote: In my experience there could be several reasons for green densities around metal atoms: 1) occupancy. In this case it is unlikely to be a problem 2) effect of f’ if it is strong. It is visible at higher resolution more than at low resolutions. 3) B vallues, isotropic or anisotropic. B value of the metal should be very similar to that of the well defined surrounding atoms 4) Multiple positions due to mixtures of charges and coordination geometries Regards Garib On 5 Sep 2025, at 12:47, Ezra Peisach <[email protected]<mailto:[email protected]>> wrote: CAUTION: This email originated from outside of the LMB: [email protected]<mailto:[email protected]>-. Do not click links or open attachments unless you recognize the sender and know the content is safe. If you think this is a phishing email, please forward it to [email protected]<mailto:[email protected]> -- I know you mentioned that this was not an anomalous map, but what at wavelength were your data collected at? Fe has a significant f" at CuKalpha (1.54..) wavelength. On 9/5/25 7:37 AM, Jon Cooper wrote: I would check the iron occupancy in case it is less than 1.0 and that your Fe has the left-shift in the PDB file otherwise it might be treated as a fluorine? Good old pdb format. Best wishes, Jon Cooper. [email protected]<mailto:[email protected]> Sent from Proton Mail Android -------- Original Message -------- On 05/09/2025 10:40, Catherine Back <[email protected]><mailto:[email protected]> wrote: Good morning, I am currently solving a structure containing four heme b molecules (res 1.7 Å), each coordinated by two histidines. The refinement is looking good, but the output from refinement has marked the Fe ions of each heme with positive density (green) in the FoFc difference map - see image. Any ideas why? I used the Monomer Library in Coot to add hemes ('HEM') in. Is it something to do with the oxidation state of the Fe? And if so, is there anything I can do about it? Best wishes, Catherine Dr Catherine R. Back (she/her) Senior Post-doctoral Research Associate School of Biochemistry University of Bristol UK ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ Confidentiality Notice: This message is private and may contain confidential and proprietary information. 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