[
{
"id": "authors:1pxvf-tsb51",
"collection": "authors",
"collection_id": "1pxvf-tsb51",
"cite_using_url": "https://authors.library.caltech.edu/records/1pxvf-tsb51",
"type": "teaching_resource",
"title": "Notes on Macromolecular Crystallography from the Turn of the Century",
"author": [
{
"family_name": "Rees",
"given_name": "Douglas C.",
"orcid": "0000-0003-4073-1185",
"clpid": "Rees-D-C"
}
],
"abstract": "I started these notes in the mid-1980s when I taught an informal course on the foundations of macromolecular crystallography for interested students and postdocs at UCLA. They were not intended as a comprehensive and systematic overview of crystallography, but rather as an introduction to some of the more mysterious or challenging aspects encountered in these studies that were of particular interest to me at that time – specifically, the relationship between X-ray scattering and structure, how to find heavy atoms and calculate phases, how to identify any non-crystallography symmetry operators, and how to refine an initial set of parameters. Phasing was generally the rate determining step in that era and so these activities naturally occupied a significant amount of the crystallographer’s time (along with preparing suitable crystals in the first place). The foundational material discussed in these notes was motivated by problems that arose in our research and the examples used to illustrate various topics were often based on these experiences. I am the first to acknowledge that many important topics, including data collection, data processing, model building and model refinement, are not discussed in the depth they merit, primarily because I was unable to improve upon the available programs and so I focused my efforts on phasing the projects under investigation in the group.
\nThese notes reflect the state of macromolecular crystallography in the early 2000s. Beginning at that time, the availability of MAD phasing methods, increasing numbers of models for molecular replacement, superb synchrotron beamlines and remarkable software packages, revolutionized the practice of macromolecular crystallography. Consequently, the ability to solve heavy atom derivatives from Patterson maps was no longer a critical survival skill. The subsequent revolutions in cryo-electron microscopy and computation (i.e., AlphaFold) have completely transformed structural biology. Not coincidentally, the driving force in structural biology has largely changed from an emphasis on solving the “first” in a family of structures, to an emphasis on the “function” part of the structure-function paradigm exemplified from the earliest days of the field by Max Perutz. As a result of these developments, there is no longer any compelling need to update the material in these notes, particularly the references, and so they reflect the state of the macromolecular crystallography at the turn of the century.
",
"doi": "10.7907/1pxvf-tsb51",
"publisher": "Caltech Library",
"publication_date": "2024-12-02"
},
{
"id": "authors:rvzkd-kt658",
"collection": "authors",
"collection_id": "rvzkd-kt658",
"cite_using_url": "https://authors.library.caltech.edu/records/rvzkd-kt658",
"type": "teaching_resource",
"title": "Problems \u2013 and Answers \u2013 in Macromolecular Crystallography",
"author": [
{
"family_name": "Rees",
"given_name": "Douglas C.",
"orcid": "0000-0003-4073-1185",
"clpid": "Rees-D-C"
}
],
"doi": "10.7907/rvzkd-kt658",
"publisher": "Caltech Library",
"publication_date": "2024-12-02"
},
{
"id": "authors:v19gb-6qa77",
"collection": "authors",
"collection_id": "v19gb-6qa77",
"cite_using_url": "https://authors.library.caltech.edu/records/v19gb-6qa77",
"type": "article",
"title": "Structural evolution of nitrogenase states under alkaline turnover",
"author": [
{
"family_name": "Warmack",
"given_name": "Rebeccah A.",
"orcid": "0000-0002-9612-0511",
"clpid": "Warmack-Rebeccah-A"
},
{
"family_name": "Rees",
"given_name": "Douglas C.",
"orcid": "0000-0003-4073-1185",
"clpid": "Rees-D-C"
}
],
"abstract": "Biological nitrogen fixation, performed by the enzyme nitrogenase, supplies nearly 50% of the bioavailable nitrogen pool on Earth, yet the structural nature of the enzyme intermediates involved in this cycle remains ambiguous. Here we present four high resolution cryoEM structures of the nitrogenase MoFe-protein, sampled along a time course of alkaline reaction mixtures under an acetylene atmosphere. This series of structures reveals a sequence of salient changes including perturbations to the inorganic framework of the FeMo-cofactor; depletion of the homocitrate moiety; diminished density around the S2B belt sulfur of the FeMo-cofactor; rearrangements of cluster-adjacent side chains; and the asymmetric displacement of the FeMo-cofactor. We further demonstrate that the nitrogenase associated factor T protein can recognize and bind an alkaline inactivated MoFe-protein in vitro. These time-resolved structures provide experimental support for the displacement of S2B and distortions of the FeMo-cofactor at the E0-E3 intermediates of the substrate reduction mechanism, prior to nitrogen binding, highlighting cluster rearrangements potentially relevant to nitrogen fixation by biological and synthetic clusters.
",
"doi": "10.1038/s41467-024-54713-0",
"pmcid": "PMC11612016",
"issn": "2041-1723",
"publisher": "Nature Publishing Group",
"publication": "Nature Communications",
"publication_date": "2024-12-02",
"series_number": "1",
"volume": "15",
"issue": "1",
"pages": "10472"
},
{
"id": "authors:zttgy-tes30",
"collection": "authors",
"collection_id": "zttgy-tes30",
"cite_using_url": "https://authors.library.caltech.edu/records/zttgy-tes30",
"type": "teaching_resource",
"title": "Crystallographic Compendium",
"author": [
{
"family_name": "Rees",
"given_name": "Douglas C.",
"orcid": "0000-0003-4073-1185",
"clpid": "Rees-D-C"
}
],
"abstract": "These notes have been prepared (and revised, re-revised, etc.) over the past decades while working through various problems encountered in our crystallographic analyses. They are not intended as a complete and systematic development of crystallography, but rather they reflect topics or problems of interest to me or that were particularly challenging that I needed to work through to better understand. This effort reflects the “learn-by-doing” approach that I have found to be valuable when I am trying to master new material.
\nThe inspiration for this compendium is Charles Kittel’s Elementary Statistical Physics (Dover, 2004, originally published in 1958) that is organized around 45 sections, each a few pages in length, that cover a set of important topics in this field. By comparison, the present effort makes no attempt (yet) to develop the material in a systematic way, either in the flow of topics, the depth of treatment, or even (alas) using a consistent set of symbols. I hope to be able to correct these defects at some time in the future. At the same time, there are always new topics to incorporate (as I learned from my time in Student Affairs “there is always more to learn”) and the evolution of this collection will undoubtedly reflect the tension between clarifying (and correcting) existing material and tackling new subjects.
",
"doi": "10.7907/zttgy-tes30",
"publisher": "Caltech Library",
"publication_date": "2024-11-26"
},
{
"id": "authors:daxtb-nwj95",
"collection": "authors",
"collection_id": "daxtb-nwj95",
"cite_using_url": "https://authors.library.caltech.edu/records/daxtb-nwj95",
"type": "article",
"title": "Analysis of early intermediate states of the nitrogenase reaction by regularization of EPR spectra",
"author": [
{
"family_name": "Heidinger",
"given_name": "Lorenz"
},
{
"family_name": "Perez",
"given_name": "Kathryn"
},
{
"family_name": "Spatzal",
"given_name": "Thomas"
},
{
"family_name": "Einsle",
"given_name": "Oliver"
},
{
"family_name": "Weber",
"given_name": "Stefan",
"orcid": "0000-0003-4090-7435"
},
{
"family_name": "Rees",
"given_name": "Douglas C.",
"orcid": "0000-0003-4073-1185",
"clpid": "Rees-D-C"
},
{
"family_name": "Schleicher",
"given_name": "Erik",
"orcid": "0000-0003-3010-9528"
}
],
"abstract": "\n
\n
Due to the complexity of the catalytic FeMo cofactor site in nitrogenases that mediates the reduction of molecular nitrogen to ammonium, mechanistic details of this reaction remain under debate. In this study, selenium- and sulfur-incorporated FeMo cofactors of the catalytic MoFe protein component from Azotobacter vinelandii are prepared under turnover conditions and investigated by using different EPR methods. Complex signal patterns are observed in the continuous wave EPR spectra of selenium-incorporated samples, which are analyzed by Tikhonov regularization, a method that has not yet been applied to high spin systems of transition metal cofactors, and by an already established grid-of-error approach. Both methods yield similar probability distributions that reveal the presence of at least four other species with different electronic structures in addition to the ground state E0. Two of these species were preliminary assigned to hydrogenated E2 states. In addition, advanced pulsed-EPR experiments are utilized to verify the incorporation of sulfur and selenium into the FeMo cofactor, and to assign hyperfine couplings of 33S and 77Se that directly couple to the FeMo cluster. With this analysis, we report selenium incorporation under turnover conditions as a straightforward approach to stabilize and analyze early intermediate states of the FeMo cofactor.
\n
\n
\n
\n
Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast majority of published cryoEM methodologies focus on the characterization of aerobically purified samples. Air-sensitive enzymes and microorganisms represent important yet understudied systems in structural biology. We have recently demonstrated the success of an anaerobic single-particle cryoEM workflow applied to the air-sensitive nitrogenase enzymes. In this protocol, we detail the use of Schlenk lines and anaerobic chambers to prepare samples, including a protein tag for monitoring sample exposure to oxygen in air. We describe how to use a plunge freezing apparatus inside of a soft-sided vinyl chamber of the type we routinely use for anaerobic biochemistry and crystallography of oxygen-sensitive proteins. Manual control of the airlock allows for introduction of liquid cryogens into the tent. A custom vacuum port provides slow, continuous evacuation of the tent atmosphere to avoid accumulation of flammable vapors within the enclosed chamber. These methods allowed us to obtain high-resolution structures of both nitrogenase proteins using single-particle cryoEM. The procedures involved can be generally subdivided into a 4 d anaerobic sample generation procedure, and a 1 d anaerobic cryoEM sample preparation step, followed by conventional cryoEM imaging and processing steps. As nitrogen is a substrate for nitrogenase, the Schlenk lines and anaerobic chambers described in this procedure are operated under an argon atmosphere; however, the system and these procedures are compatible with other controlled gas environments.
\n
\n
\n
We have examined how the refined B-factor changes as a function of Z (the atomic number of a scatterer) at the sulfur site of the [4Fe:4S] cluster of the nitrogenase iron protein by refinement. A simple model is developed that quantitatively captures the observed relationship between Z and B, based on a Gaussian electron density distribution with a constant electron density at the position of the scatterer. From this analysis, the fractional changes in B and Z are found to be similar. The utility of B-factor refinement to potentially distinguish atom types reflects the Z dependence of X-ray atomic scattering factors; the weaker dependence of electron atomic scattering factors on Z implies that distinctions between refined values of B in an electron scattering structure will be less sensitive to the atomic identity of a scatterer than for the case with X-ray-diffraction. This behavior provides an example of the complementary information that can be extracted from different types of scattering studies.
\n