Results
Selection of metalloproteins
- Kostan J.
- Sjöblom B.
- Maixner F.
- Mlynek G.
- Furtmüller P.G.
- Obinger C.
- Wagner M.
- Daims H.
- Djinović-Carugo K.

Protein | Redox potential E°′ (Fe(II)/Fe(III)) | No. of amino acids (multimer) | Secondary structure elements (%) | ||
---|---|---|---|---|---|
β-Strands | Helix | Others | |||
KpDyP | −350 ± 1 mV ( 26 ) | 299 (598) | 23.2 | 32.6 | 44 |
NdCld | −113 ± 1 mV ( 30 ) | 264 (1320) | 26.9 | 42.0 | 31 |
CCld | −126 ± 1.9 mV ( 31 ) | 182 (364) | 35.8 | 28.9 | 35.3 |
hhMb | 28 ± 5 mV ( 32 ) | 154 (154) | 0 | 73.9 | 26.1 |
AvTsdA | Heme 1 (His/Cys ligation): −185 mV ( 25 ) | 270 (270) | 4.8 | 39.5 | 55.7 |
Heme 2 (His/Lys ligation): −129 mV ( 25 ) | |||||
Heme 2 (His/Met ligation): +266 mV ( 25 ) | |||||
LmChdC | −205 ± 3 mV ( 33 ) | 251 (1255) | 31 | 37.9 | 31 |
Crystal morphology and composition

Protein | Approximate crystal volume in X-ray beam | Space group | Unit cell volume (× 10−20) | Fe(III)/unit cell | Fe(III) concentration | Fe(III) amount |
---|---|---|---|---|---|---|
μm3 | cm3 | μmol/cm3 | fmol | |||
KpDyP (6FKS) | 6200 | P 1 21 1 | 27.6 | 4 | 24.1 | 17.1 |
NdCld (3NN1) | 1570–6200 | P 32 2 1 | 250 | 30 | 19.9 | 39.3–155.3 |
CCld (5MAU) | 7850–15,700 | P 1 | 127.7 | 2 | 2.6 | 100.2–200.5 |
hhMb (1WLA) | 314 | P 1 21 1 | 6.4 | 2 | 52.0 | 0.2 |
AvTsdA (4WQ7) | 314–1570 | C 1 2 1 | 24.8 | 8 | 53.5 | 0.8–3.9 |
LmChdC (6FXJ) | 1570–3100 | P 1 21 1 | 75.4 | 10 | 22.0 | 11.8–23.4 |
Photoreduction is independent of sample-derived variables



Influence of photoreduction on resting state and ligated iron centers can be resolved using a dose-dependent method
Time/dose-resolved water-bound heme | Cyanide-bound heme | |||||||
---|---|---|---|---|---|---|---|---|
Beamline | I04 DLS | ID23-1 ESRF | ||||||
Oscillation (degrees) | 0.1 | 0.1 | ||||||
Exposure/image (s) | 0.04 | 0.1 | 0.1 | |||||
Beam size (μm) | 26 × 15 | 20 × 20 | 20 × 20 | |||||
Images/wedge | 100 | 100 | 3600 | |||||
Wavelength (Å) | 0.9795 | 0.9919 | 0.9919 | |||||
Flux (photon s−1) | 6.99 × 1010 | 1.89 × 1010 | 1.89 × 1010 | |||||
Scaled wedges | 723 | 645 | 1 | |||||
Image range from wedge | 1–4 | 5–8 | 17–20 | 35–38 | 71–74 | 96–99 | 1–5 | 1–3600 |
Dose (kGy) | 2.15 | 4.3 | 10.8 | 20.4 | 40.3 | 53.6 | 2.3 | 1590.0 |
Total exposed time (s) | 0.12 | 0.32 | 0.8 | 1.52 | 2.96 | 4 | 0.5 | 360 |
Fe(II) % reduced | 5.2 | 10.5 | 26.4 | 50 | 98.7 | 100 | 5.7 | 100 |
Space group | P 1 21 1 | P 1 21 1 | P 1 21 1 | |||||
Unit cell (Å) | a = 50.89 | a = 50.89 | a = 50.57 | |||||
b = 76.65 | b = 76.59 | b = 75.74 | ||||||
c = 76.58 | c = 76.31 | c = 75.71 | ||||||
Unit cell (degrees) | α = γ = 90.00 | α = γ = 90.00 | α = γ = 90.00 | |||||
β = 108.01 | β = 107.87 | β = 107.85 | ||||||
Resolution range (Å) | 48.40–1.90 | 48.40–1.90 | 48.40–1.90 | 47.67–1.90 | 47.67–1.90 | 47.67–1.90 | 36.31–1.80 | 36.03–1.52 |
Rmerge | 0.18 (0.48) | 0.20 (0.54) | 0.20 (0.54) | 0.20 (0.57) | 0.20 (0.58) | 0.21 (0.60) | 0.21 (0.75) | 0.06 (0.85) |
Rmeas | 0.22 (0.59) | 0.23 (0.64) | 0.24 (0.64) | 0.24 (0.68) | 0.24 (0.70) | 0.25 (0.74) | 0.25 (0.90) | 0.07 (0.10) |
Rpim | 0.12 (0.33) | 0.12 (0.34) | 0.12 (0.34) | 0.12 (0.36) | 0.13 (0.37) | 0.14 (0.41) | 0.13 (0.49) | 0.04 (0.57) |
All observations | 185,150 (11,453) | 238,147 (14,639) | 241,170 (14,925) | 241,538 (14,758) | 235,160 (14,352) | 202,833 (12,376) | 267,452 (15,345) | 246,785 (24,852) |
Unique observations | 42,694 (2727) | 43,499 (2791) | 43,564 (2794) | 43,584 (2795) | 43,552 (2789) | 43,298 (2778) | 51,235 (3003) | 81,528 (8142) |
Mean (I/σI) | 5.4 (3.0) | 6.2 (3.4) | 6.1 (3.4) | 6.2 (3.4) | 6.0 (3.3) | 5.5 (2.9) | 6.1 (2.7) | 3.0 (3.1) |
CC½ | 0.96 (0.65) | 0.96 (0.37) | 0.96 (0.47) | 0.96 (0.39) | 0.95 (0.33) | 0.95 (0.41) | 0.97 (0.70) | 0.99 (0.50) |
Completeness (%) | 96.8 (96.6) | 98.6 (98.8) | 98.7 (98.9) | 98.8 (98.9) | 98.7 (98.7) | 98.1 (98.3) | 99.2 (99.0) | 97.8 (97.4) |
Multiplicity | 4.3 (4.2) | 5.5 (5.2) | 5.5 (5.3) | 5.5 (5.3) | 5.4 (5.1) | 4.7 (4.5) | 5.2 (5.1) | 3.0 (3.1) |
Rwork (%) | 17.2 | 17 | 16.9 | 18.4 | 18.1 | 17.2 | 17.2 | 16.7 |
Rfree (%) | 19.9 | 19.1 | 18.1 | 20.1 | 19.8 | 19 | 19.7 | 18.2 |
Refined residues | 599 | 599 | 599 | 599 | 599 | 599 | 597 | 597 |
Refined H2O | 671 | 634 | 655 | 644 | 621 | 669 | 785 | 560 |
Average B-factor (Å2) | 12 | 12 | 12 | 12 | 12 | 12 | 10.34 | 26.26 |
RMSD bond length (Å) | 0.023 | 0.022 | 0.024 | 0.018 | 0.017 | 0.015 | 0.013 | 0.015 |
RMSD bond angle (degrees) | 1.54 | 1.52 | 1.59 | 1.42 | 1.36 | 1.24 | 1.15 | 1.66 |
Ramachandran favored (%) | 98.15 | 97.98 | 97.64 | 98.15 | 97.98 | 97.82 | 98.65 | 98.48 |
Rotamer outliers (%) | 0.6 | 1.18 | 1.19 | 0.8 | 1.01 | 0.4 | 1.38 | 1.2 |
Molprobity overall (percentile) | 100 | 98 | 99 | 100 | 100 | 100 | 99 | 100 |
PDB accession code | 6RQY | 6RR1 | 6RR4 | 6RR5 | 6RR6 | 6RR8 | 6RPE | 6RPD |


Discussion
Experimental procedures
Protein preparation and crystallization conditions
Online UV-visible spectroscopy and beamline settings
UV-visible spectroscopy of ferric and ferrous heme proteins in solution
X-ray data collection and structure determination
Data reduction
Phasing and model building
Data availability
Acknowledgments
Supplementary Material
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Article info
Publication history
Footnotes
This article contains supporting information.
Author contributions—V. P., K. D.-C., and S. H. conceptualization; V. P., J. H. B., H. M., D. S., T. G., and S. H. data curation; V. P., J. H. B., H. M., D. S., T. G., and S. H. formal analysis; V. P. and S. H. supervision; V. P., J. H. B., and S. H. validation; V. P. and S. H. visualization; V. P., J. H. B., K. D.-C., and S. H. methodology; V. P.,J. H. B., K. D.-C., and S. H. writing-original draft; V. P. and S. H. project administration; V. P., J. H. B., C. O., K. D.-C., and S. H. writing-review and editing; J. H. B. software; C. O. and K. D.-C. resources; C. O., K. D.-C., and S. H. funding acquisition.
Funding and additional information—This project was supported by the Austrian Science Fund, FWF (Doctoral Program BioToP - Molecular Technology of Proteins (W1224) and the project P29099), Diamond Light Source Proposal MX19036, ESRF Proposal MX1998, and the iNEXT program (PID: 4482). The work of K. D.-C. was supported by Austrian Science Fund (FWF) Projects I525, I1593, P22276, P19060, and W1221; Wellcome Trust Collaborative Award 201543/Z/16/Z; by COST action BM1405—Non-globular proteins–from sequence to structure, function, and application in molecular physiopathology (NGP-NET); WWTF (Vienna Science and Technology Fund) Chemical Biology Project LS17-008; the Austrian Federal Ministry of Science, Research, and Economy; and the National Foundation for Research, Technology, and Development through funding the Christian Doppler Laboratory for High-Content Structural Biology and Biotechnology, Austria.
Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.
Present address for John H. Beale: Paul Scherrer Institute, Villigen, Switzerland.
Abbreviations—The abbreviations used are: Gy
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