Thank you Frances and Chris, yes the F in Fe is left-set in the PDB file, but a good thing to check!
Dr Catherine R. Back (she/her) Senior Post-doctoral Research Associate School of Biochemistry University of Bristol UK [email protected] ________________________________ From: CCP4 bulletin board <[email protected]> on behalf of Frances C. Bernstein <[email protected]> Sent: 05 September 2025 16:49 To: [email protected] <[email protected]> Subject: Re: [ccp4bb] Green density over Fe in heme A little bit of history here about PDB format atom names. At the start of the PDB, the format was 140 columns wide based on the output of the Diamond real-space refinement program. This was used for the first 100 entries but we realized that we needed to go to a more compact format. That yielded what is called the PDB format and the first 100 entires were converted to PDB format. The atom name field is 4 columns wide. The first 2 are the chemical element, right-justified and the second 2 are a position indicator. This is why it is important to correctly position the element name if it is 2 characters long, e.g. FE; namely it must be in the first 2 columns of the 4. This was over 50 years ago and thanks to modern medicine all of us that were involved in this are still alive. I am the only one, however, that follows this list so I decided to explain this aspect of the PDB format that we designed back then. Frances Bernstein On 2025-09-05 11:31, Colbert, Christopher wrote: Hi all, One thing I haven't seen mentioned is to be sure that the F for the Fe atom is in the correct column of the pdb file. Metals are left set from C, N, O, S, CA, CB, etc. Cheers, Chris -- Christopher L. Colbert, Ph.D. Pronouns: he/him/his Professor Department of Chemistry and Biochemistry NORTH DAKOTA STATE UNIVERSITY QBB 346 Dept 2710, P.O. Box 6050 Fargo, ND 58108-6050 phone: 701.231.7946 fax: 701.231.8324 [email protected]<mailto:[email protected]> From: CCP4 bulletin board <[email protected]> on behalf of Oganesyan, Vaheh <[email protected]> Date: Friday, September 5, 2025 at 8:24 AM To: [email protected] <[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 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]> On Behalf Of Catherine Back Sent: Friday, September 5, 2025 8:51 AM To: [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. 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