Special Issue "Recent Advances in Protein Crystallography"

A special issue of Crystals (ISSN 2073-4352). This special issue belongs to the section "Biomolecular Crystals".

Deadline for manuscript submissions: closed (31 December 2017)

Special Issue Editor

Guest Editor
Dr. Albert Guskov

Faculty of Mathematics and Natural Sciences, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
Website | E-Mail
Interests: membrane proteins; protein crystallography; protein crystallization

Special Issue Information

Dear Colleagues,

The field of the structural biology is thriving owing to several so-called “revolutions”, such as the advent of X-ray Free Electron Lasers, remarkable improvements in detectors’ resolution for Cryo-electron Microscopy and many other important developments in all aspects of the field: Protein expression, purification, crystallization, data processing and analysis, and so on. We decided to compile this Special Issue on “Recent advances in Protein Crystallography” to summarize the current progress and the state-of-the-art of the structural biology. Authors are encouraged to submit their manuscripts covering topics expressed in the keywords below. The additional goal of this issue is to provide the growing community of scientists interested in structural biology with an excellent reference material. Additionally we will compile the subsection on protein structures, thus manuscripts describing new structures with the structural-functional analysis or “old” structures but with a new twist are also welcome.

Dr. Albert Guskov
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All papers will be peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Crystals is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1200 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Structural biology

  • Protein crystallography

  • Membrane proteins

  • Protein crystallization

  • X-ray Free Electron Laser

  • Cryo-Electron Microscopy

  • Protein Expression and purification

  • Data processing and analysis

  • Refinement and validation

  • Biophysical characterization

Published Papers (13 papers)

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Research

Jump to: Review

Open AccessArticle Resolution Dependence of an Ab Initio Phasing Method in Protein X-ray Crystallography
Crystals 2018, 8(4), 156; https://doi.org/10.3390/cryst8040156
Received: 17 February 2018 / Revised: 30 March 2018 / Accepted: 31 March 2018 / Published: 3 April 2018
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Abstract
For direct phasing of protein crystals, a method based on the hybrid-input-output (HIO) algorithm has been proposed and tested on a variety of structures. So far, however, the diffraction data have been limited to high-resolution ones, i.e., higher than 2 Å. In principle,
[...] Read more.
For direct phasing of protein crystals, a method based on the hybrid-input-output (HIO) algorithm has been proposed and tested on a variety of structures. So far, however, the diffraction data have been limited to high-resolution ones, i.e., higher than 2 Å. In principle, the methodology can be applied to data of lower resolutions, which might be particularly useful for phasing membrane protein crystals. For resolutions higher than 3.5 Å, it seems the atomic structure is solvable. For data of lower resolutions, information of the secondary structures and the protein boundary can still be obtained. Examples are given to support the conclusions. Real experimental data are used. Two aspects of the observed data have been discussed: removal of the measured low-resolution reflections and involvement of the unmeasured high-resolution reflections. The ab initio phasing employs histogram matching for density modification. A question arises whether the reference histogram used should match the resolution of the diffraction data or not. It seems that there is an optimal histogram which is good to use for data at various resolutions. Full article
(This article belongs to the Special Issue Recent Advances in Protein Crystallography)
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Open AccessArticle Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS)
Crystals 2018, 8(3), 109; https://doi.org/10.3390/cryst8030109
Received: 31 January 2018 / Revised: 22 February 2018 / Accepted: 22 February 2018 / Published: 26 February 2018
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Abstract
The dynamism of proteins is central to their function, and several proteins have been described as flexible, as consisting of multiple domains joined by flexible linkers, and even as intrinsically disordered. Several techniques exist to study protein structures, but small angle X-ray scattering
[...] Read more.
The dynamism of proteins is central to their function, and several proteins have been described as flexible, as consisting of multiple domains joined by flexible linkers, and even as intrinsically disordered. Several techniques exist to study protein structures, but small angle X-ray scattering (SAXS) has proven to be particularly powerful for the quantitative analysis of such flexible systems. In the present report, we have used SAXS in combination with X-ray crystallography to highlight their usefulness at characterizing flexible proteins, using as examples two proteins involved in different steps of ribosome biogenesis. The yeast BRCA2 and CDKN1A-interactig protein, Bcp1, is a chaperone for Rpl23 of unknown structure. We showed that it consists of a rigid, slightly elongated protein, with a secondary structure comprising a mixture of alpha helices and beta sheets. As an example of a flexible molecule, we studied the SBDS (Shwachman-Bodian-Diamond Syndrome) protein that is involved in the cytoplasmic maturation of the 60S subunit and constitutes the mutated target in the Shwachman-Diamond Syndrome. In solution, this protein coexists in an ensemble of three main conformations, with the N- and C-terminal ends adopting different orientations with respect to the central domain. The structure observed in the protein crystal corresponds to an average of those predicted by the SAXS flexibility analysis. Full article
(This article belongs to the Special Issue Recent Advances in Protein Crystallography)
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Open AccessArticle Crystal Structure of Shigella flexneri SF173 Reveals a Dimeric Helical Bundle Conformation
Crystals 2018, 8(2), 97; https://doi.org/10.3390/cryst8020097
Received: 29 December 2017 / Revised: 12 February 2018 / Accepted: 12 February 2018 / Published: 14 February 2018
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Abstract
We report the crystal structure and bioinformatic analysis of SF173, a functionally uncharacterized protein from the human enteropathogenic bacteria Shigella flexneri. The structure shows a tightly interlinked dimer formed by adimeric core comprising α2 and α3 helices from both subunits and swapping
[...] Read more.
We report the crystal structure and bioinformatic analysis of SF173, a functionally uncharacterized protein from the human enteropathogenic bacteria Shigella flexneri. The structure shows a tightly interlinked dimer formed by adimeric core comprising α2 and α3 helices from both subunits and swapping the N-terminal α1 helix of each monomer. Structural inspection and genomic analysis results suggest that the SF173 might play its putative function by binding to SF172, the partially overlapped upstream product in the operon. As YaeO (an SF172 orthologue) has been identified to be an inhibitor of the bacterial transcription terminator Rho protein, SF173 is suggested to be involved in the regulation of Rho-dependent transcription termination, by inhibiting the Rho protein binding to SF172/YaeO. Full article
(This article belongs to the Special Issue Recent Advances in Protein Crystallography)
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Open AccessArticle Characterization of the PB2 Cap Binding Domain Accelerates Inhibitor Design
Crystals 2018, 8(2), 62; https://doi.org/10.3390/cryst8020062
Received: 7 December 2017 / Revised: 16 January 2018 / Accepted: 23 January 2018 / Published: 28 January 2018
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Abstract
X-ray crystallographic structural determinations of the PB2 cap binding domain (PB2cap) have improved the conformational characterization of the RNA-dependent RNA polymerase machinery (PA, PB2, and PB1) of the influenza virus. Geometrically, the catalytic PB1 subunit resembles the palm of a human hand. PA
[...] Read more.
X-ray crystallographic structural determinations of the PB2 cap binding domain (PB2cap) have improved the conformational characterization of the RNA-dependent RNA polymerase machinery (PA, PB2, and PB1) of the influenza virus. Geometrically, the catalytic PB1 subunit resembles the palm of a human hand. PA lies near the thumb region, and PB2 lies near the finger region. PB2 binds the cap moiety in the pre-mRNA of the host cell, while the endonuclease of PA cleaves the pre-mRNA 10–13 nucleotides downstream. The truncated RNA piece performs as a primer for PB1 to synthesize the viral mRNA. Precisely targeting PB2cap with a small molecule inhibitor will halt viral proliferation via interference of the cap-snatching activity. Wild-type and mutant PB2cap from A/California/07/2009 H1N1 were expressed in Escherichia coli, purified by nickel affinity and size exclusion chromatography, crystallized, and subjected to X-ray diffraction experiments. The crystal of mutant PB2cap liganded with m7GTP was prepared by co-crystallization. Structures were solved by the molecular replacement method, refined, and deposited in the Protein Data Bank (PDB). Structural determination and comparative analyses of these structures revealed the functions of Glu361, Lys376, His357, Phe404, Phe323, Lys339, His432, Asn429, Gln406, and Met401 in PB2cap, and the dissociation of the influenza A PB2cap C-terminal subdomain (residues 446–479) upon ligand binding. Understanding the role of these residues will aid in the ultimate development of a small-molecule inhibitor that binds both Influenza A and B virus PB2cap. Full article
(This article belongs to the Special Issue Recent Advances in Protein Crystallography)
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Open AccessArticle High-Resolution Crystal Structure of RpoS Fragment including a Partial Region 1.2 and Region 2 from the Intracellular Pathogen Legionella pneumophila
Crystals 2018, 8(2), 54; https://doi.org/10.3390/cryst8020054
Received: 25 November 2017 / Revised: 17 January 2018 / Accepted: 20 January 2018 / Published: 23 January 2018
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Abstract
Legionella pneumophila RpoS (LpRpoS), an alternative sigma factor of RNA polymerase (RNAP), is essential for virulence and stress resistance. To investigate the mechanism of RpoS in the intracellular pathogen L. pneumophila, we determined the high-resolution crystal structure of the Lp
[...] Read more.
Legionella pneumophila RpoS (LpRpoS), an alternative sigma factor of RNA polymerase (RNAP), is essential for virulence and stress resistance. To investigate the mechanism of RpoS in the intracellular pathogen L. pneumophila, we determined the high-resolution crystal structure of the LpRpoS 95–195 containing a partial region 1.2 and region 2. The structure of LpRpoS 95–195 reveals that the conserved residues are critical for promoter melting, DNA and core RNAP binding. The differences in regulatory factor binding site between Escherichia coli RpoS and LpRpoS suggest that LpRpoS may employ a distinct mechanism to recruit alternative regulatory factors controlling transcription initiation. Full article
(This article belongs to the Special Issue Recent Advances in Protein Crystallography)
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Open AccessArticle Identification and Crystallographic Analysis of a New Carbohydrate Acetylesterase (SmAcE1) from Sinorhizobium meliloti
Crystals 2018, 8(1), 12; https://doi.org/10.3390/cryst8010012
Received: 23 October 2017 / Revised: 20 December 2017 / Accepted: 24 December 2017 / Published: 1 January 2018
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Abstract
Carbohydrate-active enzymes (CAZymes) regulate the synthesis, degradation, and modification of the poly—and oligosaccharides in all three kingdoms of life. A novel carbohydrate acetylesterase from Sinorhizobium meliloti, designated SmAcE1, was identified, characterized, and crystallized. This SmAcE1 is classified into the carbohydrate esterase family
[...] Read more.
Carbohydrate-active enzymes (CAZymes) regulate the synthesis, degradation, and modification of the poly—and oligosaccharides in all three kingdoms of life. A novel carbohydrate acetylesterase from Sinorhizobium meliloti, designated SmAcE1, was identified, characterized, and crystallized. This SmAcE1 is classified into the carbohydrate esterase family 3 (CE3) based on the sequence alignments with other currently known carbohydrate esterase (CE) family enzymes. The SmAcE1 was crystallized as a hexamer in a space group P212121 with the unit cell parameters: a = 99.12 Å, b = 148.88 Å, c = 149.84 Å, and α = β = γ = 90.00°. The diffraction data set was collected up to a 2.05 Å resolution. Hydrolysis activity of SmAcE1 towards glucose pentaacetate and cellulose acetate was further confirmed using acetic acid release assay. Further crystallographic and functional analyses studies on SmAcE1 would be followed to fully understand the reaction mechanisms of CEs. Full article
(This article belongs to the Special Issue Recent Advances in Protein Crystallography)
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Open AccessArticle Expression, Purification, Crystallization, and X-ray Structural Analysis of CRISPR-Associated Protein Cas6 from Methanocaldococcus jannaschii
Crystals 2017, 7(11), 344; https://doi.org/10.3390/cryst7110344
Received: 20 September 2017 / Revised: 7 November 2017 / Accepted: 8 November 2017 / Published: 10 November 2017
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Abstract
The CRISPR-associated protein 6, Cas6 protein, is an endoribonuclease that cleaves precursor CRISPR RNAs within the repeat sequence to release specific invader-targeting RNAs. Cas6 protein can recognize different sequences by their specific scaffold. To investigate its binding mode, we purified and crystallized a
[...] Read more.
The CRISPR-associated protein 6, Cas6 protein, is an endoribonuclease that cleaves precursor CRISPR RNAs within the repeat sequence to release specific invader-targeting RNAs. Cas6 protein can recognize different sequences by their specific scaffold. To investigate its binding mode, we purified and crystallized a His-tagged Cas6 protein from Methanocaldococcus jannaschii (MjCas6) using the sitting-drop vapor-diffusion method. The crystals diffracted to a resolution of 1.85 Å and belonged to monoclinic space group C2, with unit-cell parameters a = 200.84 Å, b = 85.26 Å, c = 100.06 Å, β = 118.47°. The crystals of MjCas6 contain four molecules in the asymmetric unit. The protein fold is similar to the other Cas6 homologues, such as Pyrococcus furiosus Cas6, suggesting functional similarity. Moreover, in the C2 crystal the MjCas6 monomers formed a tandem array, which we hypothesize to possibly correlate with repetitive RNA precursors. Full article
(This article belongs to the Special Issue Recent Advances in Protein Crystallography)
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Open AccessArticle The Use of Size Exclusion Chromatography to Monitor Protein Self-Assembly
Crystals 2017, 7(11), 331; https://doi.org/10.3390/cryst7110331
Received: 6 September 2017 / Revised: 20 October 2017 / Accepted: 23 October 2017 / Published: 31 October 2017
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Abstract
High resolution size exclusion chromatography (SEC) coupled with static light scattering (SLS) analyses were conducted to study the effect of the mobile phase ionic strength and protein concentration on the output of SEC experiments. The results highlight the effect of small changes in
[...] Read more.
High resolution size exclusion chromatography (SEC) coupled with static light scattering (SLS) analyses were conducted to study the effect of the mobile phase ionic strength and protein concentration on the output of SEC experiments. The results highlight the effect of small changes in the mobile phase composition on the estimation of molar masses estimated from retention time-based calibration curve compared with those obtained from SLS analysis. By comparing the SLS data with the SEC chromatograms, we show that SEC can provide helpful information on the protein aggregation state as macromolecules approach known precipitation points in their phase diagrams. This suggests the potential use of SEC as an easily accessible lab-based scanning methodology to monitor protein self-assembly prior to nucleation and crystallization. Implications for the use of SEC to study protein phase diagrams are discussed. Full article
(This article belongs to the Special Issue Recent Advances in Protein Crystallography)
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Open AccessArticle Crystal Structure of the 23S rRNA Fragment Specific to r-Protein L1 and Designed Model of the Ribosomal L1 Stalk from Haloarcula marismortui
Crystals 2017, 7(2), 37; https://doi.org/10.3390/cryst7020037
Received: 13 December 2016 / Accepted: 29 January 2017 / Published: 2 February 2017
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Abstract
The crystal structure of the 92-nucleotide L1-specific fragment of 23S rRNA from Haloarcula marismortui (Hma) has been determined at 3.3 Å resolution. Similar to the corresponding bacterial rRNA fragments, this structure contains joined helix 76-77 topped by an approximately globular structure formed by
[...] Read more.
The crystal structure of the 92-nucleotide L1-specific fragment of 23S rRNA from Haloarcula marismortui (Hma) has been determined at 3.3 Å resolution. Similar to the corresponding bacterial rRNA fragments, this structure contains joined helix 76-77 topped by an approximately globular structure formed by the residual part of the L1 stalk rRNA. The position of HmaL1 relative to the rRNA was found by its docking to the rRNA fragment using the L1-rRNA complex from Thermus thermophilus as a guide model. In spite of the anomalous negative charge of the halophilic archaeal protein, the conformation of the HmaL1-rRNA interface appeared to be very close to that observed in all known L1-rRNA complexes. The designed structure of the L1 stalk was incorporated into the H. marismortui 50S ribosomal subunit. Comparison of relative positions of L1 stalks in 50S subunits from H. marismortui and T. thermophilus made it possible to reveal the site of inflection of rRNA during the ribosome function. Full article
(This article belongs to the Special Issue Recent Advances in Protein Crystallography)
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Open AccessArticle Crystal Structure of the Substrate-Binding Domain from Listeria monocytogenes Bile-Resistance Determinant BilE
Crystals 2016, 6(12), 162; https://doi.org/10.3390/cryst6120162
Received: 28 October 2016 / Revised: 29 November 2016 / Accepted: 6 December 2016 / Published: 9 December 2016
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Abstract
BilE has been reported as a bile resistance determinant that plays an important role in colonization of the gastrointestinal tract by Listeria monocytogenes, the causative agent of listeriosis. The mechanism(s) by which BilE mediates bile resistance are unknown. BilE shares significant sequence
[...] Read more.
BilE has been reported as a bile resistance determinant that plays an important role in colonization of the gastrointestinal tract by Listeria monocytogenes, the causative agent of listeriosis. The mechanism(s) by which BilE mediates bile resistance are unknown. BilE shares significant sequence similarity with ATP-binding cassette (ABC) importers that contribute to virulence and stress responses by importing quaternary ammonium compounds that act as compatible solutes. Assays using related compounds have failed to demonstrate transport mediated by BilE. The putative substrate-binding domain (SBD) of BilE was expressed in isolation and the crystal structure solved at 1.5 Å. Although the overall fold is characteristic of SBDs, the binding site varies considerably relative to the well-characterized homologs ProX from Archaeoglobus fulgidus and OpuBC and OpuCC from Bacillus subtilis. This suggests that BilE may bind an as-yet unknown ligand. Elucidation of the natural substrate of BilE could reveal a novel bile resistance mechanism. Full article
(This article belongs to the Special Issue Recent Advances in Protein Crystallography)
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Review

Jump to: Research

Open AccessReview Radiation Damage in Macromolecular Crystallography—An Experimentalist’s View
Crystals 2018, 8(4), 157; https://doi.org/10.3390/cryst8040157
Received: 5 March 2018 / Revised: 22 March 2018 / Accepted: 24 March 2018 / Published: 4 April 2018
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Abstract
Radiation damage still remains a major limitation and challenge in macromolecular X-ray crystallography. Some of the high-intensity radiation used for diffraction data collection experiments is absorbed by the crystals, generating free radicals. These give rise to radiation damage even at cryotemperatures (~100 K),
[...] Read more.
Radiation damage still remains a major limitation and challenge in macromolecular X-ray crystallography. Some of the high-intensity radiation used for diffraction data collection experiments is absorbed by the crystals, generating free radicals. These give rise to radiation damage even at cryotemperatures (~100 K), which can lead to incorrect biological conclusions being drawn from the resulting structure, or even prevent structure solution entirely. Investigation of mitigation strategies and the effects caused by radiation damage has been extensive over the past fifteen years. Here, recent understanding of the physical and chemical phenomena of radiation damage is described, along with the global effects inflicted on the collected data and the specific effects observed in the solved structure. Furthermore, this review aims to summarise the progress made in radiation damage studies in macromolecular crystallography from the experimentalist’s point of view and to give an introduction to the current literature. Full article
(This article belongs to the Special Issue Recent Advances in Protein Crystallography)
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Open AccessReview An Overview of the Top Ten Detergents Used for Membrane Protein Crystallization
Crystals 2017, 7(7), 197; https://doi.org/10.3390/cryst7070197
Received: 7 June 2017 / Revised: 26 June 2017 / Accepted: 28 June 2017 / Published: 1 July 2017
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Abstract
To study integral membrane proteins, one has to extract them from the membrane—the step that is typically achieved by the application of detergents. In this mini-review, we summarize the top 10 detergents used for the structural analysis of membrane proteins based on the
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To study integral membrane proteins, one has to extract them from the membrane—the step that is typically achieved by the application of detergents. In this mini-review, we summarize the top 10 detergents used for the structural analysis of membrane proteins based on the published results. The aim of this study is to provide the reader with an overview of the main properties of available detergents (critical micelle concentration (CMC) value, micelle size, etc.) and provide an idea of what detergents to may merit further study. Furthermore, we briefly discuss alternative solubilization and stabilization agents, such as polymers. Full article
(This article belongs to the Special Issue Recent Advances in Protein Crystallography)
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Open AccessReview The Synergetic Effects of Combining Structural Biology and EPR Spectroscopy on Membrane Proteins
Crystals 2017, 7(4), 117; https://doi.org/10.3390/cryst7040117
Received: 14 February 2017 / Revised: 9 April 2017 / Accepted: 12 April 2017 / Published: 20 April 2017
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Abstract
Protein structures as provided by structural biology such as X-ray crystallography, cryo-electron microscopy and NMR spectroscopy are key elements to understand the function of a protein on the molecular level. Nonetheless, they might be error-prone due to crystallization artifacts or, in particular in
[...] Read more.
Protein structures as provided by structural biology such as X-ray crystallography, cryo-electron microscopy and NMR spectroscopy are key elements to understand the function of a protein on the molecular level. Nonetheless, they might be error-prone due to crystallization artifacts or, in particular in case of membrane-imbedded proteins, a mostly artificial environment. In this review, we will introduce different EPR spectroscopy methods as powerful tools to complement and validate structural data gaining insights in the dynamics of proteins and protein complexes such that functional cycles can be derived. We will highlight the use of EPR spectroscopy on membrane-embedded proteins and protein complexes ranging from receptors to secondary active transporters as structural information is still limited in this field and the lipid environment is a particular challenge. Full article
(This article belongs to the Special Issue Recent Advances in Protein Crystallography)
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