Role of Computational Methods in Going beyond X-ray Crystallography to Explore Protein Structure and Dynamics

Protein structural biology came a long way since the determination of the first three-dimensional structure of myoglobin about six decades ago. Across this period, X-ray crystallography was the most important experimental method for gaining atomic-resolution insight into protein structures. However, as the role of dynamics gained importance in the function of proteins, the limitations of X-ray crystallography in not being able to capture dynamics came to the forefront. Computational methods proved to be immensely successful in understanding protein dynamics in solution, and they continue to improve in terms of both the scale and the types of systems that can be studied. In this review, we briefly discuss the limitations of X-ray crystallography in studying protein dynamics, and then provide an overview of different computational methods that are instrumental in understanding the dynamics of proteins and biomacromolecular complexes.

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A new on-axis multimode spectrometer for the macromolecular crystallography beamlines of the Swiss Light Source

Journal of Synchrotron Radiation ◽

10.1107/s0909049508040120 ◽

2009 ◽

Vol 16 (2) ◽

pp. 173-182 ◽

Cited By ~ 34

Author(s):

Robin L. Owen ◽

Arwen R. Pearson ◽

Alke Meents ◽

Pirmin Boehler ◽

Vincent Thominet ◽

...

Keyword(s):

Light Source ◽

Three Dimensional ◽

Dimensional Structure ◽

X Ray ◽

X Ray Crystallography ◽

Additional Information ◽

Swiss Light Source ◽

Induced Changes ◽

Insight Into

X-ray crystallography at third-generation synchrotron sources permits tremendous insight into the three-dimensional structure of macromolecules. Additional information is, however, often required to aid the transition from structure to function. In situ spectroscopic methods such as UV–Vis absorption and (resonance) Raman can provide this, and can also provide a means of detecting X-ray-induced changes. Here, preliminary results are introduced from an on-axis UV–Vis absorption and Raman multimode spectrometer currently being integrated into the beamline environment at X10SA of the Swiss Light Source. The continuing development of the spectrometer is also outlined.

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History of protein crystallography in China

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2007.2032 ◽

2007 ◽

Vol 362 (1482) ◽

pp. 1035-1042 ◽

Cited By ~ 3

Author(s):

Zihe Rao

Keyword(s):

Protein Crystallography ◽

Three Dimensional ◽

Porcine Insulin ◽

Dimensional Structure ◽

X Ray Diffraction ◽

X Ray ◽

X Ray Crystallography ◽

Subsequent Determination ◽

History Of

China has a strong background in X-ray crystallography dating back to the 1920s. Protein crystallography research in China was first developed following the successful synthesis of insulin in China in 1966. The subsequent determination of the three-dimensional structure of porcine insulin made China one of the few countries which could determine macromolecular structures by X-ray diffraction methods in the late 1960s and early 1970s. After a slow period during the 1970s and 1980s, protein crystallography in China has reached a new climax with a number of outstanding accomplishments. Here, I review the history and progress of protein crystallography in China and detail some of the recent research highlights, including the crystal structures of two membrane proteins as well as the structural genomics initiative in China.

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Membrane protein crystallography in the era of modern structural biology

Biochemical Society Transactions ◽

10.1042/bst20200066 ◽

2020 ◽

Vol 48 (6) ◽

pp. 2505-2524

Author(s):

Tristan O. C. Kwan ◽

Danny Axford ◽

Isabel Moraes

Keyword(s):

Structural Biology ◽

Molecular Level ◽

Protein Structures ◽

Three Dimensional ◽

Biological Macromolecules ◽

X Ray ◽

X Ray Crystallography ◽

Cellular Processes ◽

Biochemical Level

The aim of structural biology has been always the study of biological macromolecules structures and their mechanistic behaviour at molecular level. To achieve its goal, multiple biophysical methods and approaches have become part of the structural biology toolbox. Considered as one of the pillars of structural biology, X-ray crystallography has been the most successful method for solving three-dimensional protein structures at atomic level to date. It is however limited by the success in obtaining well-ordered protein crystals that diffract at high resolution. This is especially true for challenging targets such as membrane proteins (MPs). Understanding structure-function relationships of MPs at the biochemical level is vital for medicine and drug discovery as they play critical roles in many cellular processes. Though difficult, structure determination of MPs by X-ray crystallography has significantly improved in the last two decades, mainly due to many relevant technological and methodological developments. Today, numerous MP crystal structures have been solved, revealing many of their mechanisms of action. Yet the field of structural biology has also been through significant technological breakthroughs in recent years, particularly in the fields of single particle electron microscopy (cryo-EM) and X-ray free electron lasers (XFELs). Here we summarise the most important advancements in the field of MP crystallography and the significance of these developments in the present era of modern structural biology.

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Determination of the Three-dimensional Structure of Dislocations by Synchrotron White X-ray Topography Combined with a Topo-tomographic Technique

Materia Japan ◽

10.2320/materia.46.823 ◽

2007 ◽

Vol 46 (12) ◽

pp. 823-823

Author(s):

Seiji Kawado ◽

Toshinori Taishi ◽

Satoshi Iida ◽

Yoshifumi Suzuki ◽

Yoshinori Chikaura ◽

...

Keyword(s):

Three Dimensional ◽

Dimensional Structure ◽

X Ray ◽

Three Dimensional Structure ◽

Tomographic Technique

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MRPC (Missing Regions in Polypeptide Chains): a knowledgebase

Journal of Applied Crystallography ◽

10.1107/s1600576719012330 ◽

2019 ◽

Vol 52 (6) ◽

pp. 1422-1426

Author(s):

Rajendran Santhosh ◽

Namrata Bankoti ◽

Adgonda Malgonnavar Padmashri ◽

Daliah Michael ◽

Jeyaraman Jeyakanthan ◽

...

Keyword(s):

Protein Structures ◽

Three Dimensional ◽

Protein Molecule ◽

Data Bank ◽

Protein Crystal ◽

Dimensional Structure ◽

Protein Structure Analysis ◽

Three Dimensional Structure ◽

X Ray Crystallography ◽

Polypeptide Chains

Missing regions in protein crystal structures are those regions that cannot be resolved, mainly owing to poor electron density (if the three-dimensional structure was solved using X-ray crystallography). These missing regions are known to have high B factors and could represent loops with a possibility of being part of an active site of the protein molecule. Thus, they are likely to provide valuable information and play a crucial role in the design of inhibitors and drugs and in protein structure analysis. In view of this, an online database, Missing Regions in Polypeptide Chains (MRPC), has been developed which provides information about the missing regions in protein structures available in the Protein Data Bank. In addition, the new database has an option for users to obtain the above data for non-homologous protein structures (25 and 90%). A user-friendly graphical interface with various options has been incorporated, with a provision to view the three-dimensional structure of the protein along with the missing regions using JSmol. The MRPC database is updated regularly (currently once every three months) and can be accessed freely at the URL http://cluster.physics.iisc.ac.in/mrpc.

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The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism

Biochemical Journal ◽

10.1042/bj3330811 ◽

1998 ◽

Vol 333 (3) ◽

pp. 811-816 ◽

Cited By ~ 14

Author(s):

Antonio PÁRRAGA ◽

Isabel GARCÍA-SÁEZ ◽

Sinead B. WALSH ◽

Timothy J. MANTLE ◽

Miquel COLL

Keyword(s):

Conformational Changes ◽

Active Site ◽

Three Dimensional ◽

Dimensional Structure ◽

Water Molecules ◽

X Ray Diffraction ◽

Glutathione S Transferase ◽

X Ray ◽

The Right

The structure of mouse liver glutathione S-transferase P1-1 complexed with its substrate glutathione (GSH) has been determined by X-ray diffraction analysis. No conformational changes in the glutathione moiety or in the protein, other than small adjustments of some side chains, are observed when compared with glutathione adduct complexes. Our structure confirms that the role of Tyr-7 is to stabilize the thiolate by hydrogen bonding and to position it in the right orientation. A comparison of the enzyme–GSH structure reported here with previously described structures reveals rearrangements in a well-defined network of water molecules in the active site. One of these water molecules (W0), identified in the unliganded enzyme (carboxymethylated at Cys-47), is displaced by the binding of GSH, and a further water molecule (W4) is displaced following the binding of the electrophilic substrate and the formation of the glutathione conjugate. The possibility that one of these water molecules participates in the proton abstraction from the glutathione thiol is discussed.

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Topography Prediction of Helical Transmembrane Proteins by a New Modification of the Sliding Window Method

BioMed Research International ◽

10.1155/2014/921218 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Maria N. Simakova ◽

Nikolai N. Simakov

Keyword(s):

Membrane Proteins ◽

Transmembrane Domain ◽

Domain Boundary ◽

Three Dimensional ◽

Transmembrane Proteins ◽

Sliding Window ◽

Dimensional Structure ◽

X Ray ◽

X Ray Crystallography ◽

Window Method

Protein functions are specified by its three-dimensional structure, which is usually obtained by X-ray crystallography. Due to difficulty of handling membrane proteins experimentally to date the structure has only been determined for a very limited part of membrane proteins (<4%). Nevertheless, investigation of structure and functions of membrane proteins is important for medicine and pharmacology and, therefore, is of significant interest. Methods of computer modeling based on the data on the primary protein structure or the symbolic amino acid sequence have become an actual alternative to the experimental method of X-ray crystallography for investigating the structure of membrane proteins. Here we presented the results of the study of 35 transmembrane proteins, mainly GPCRs, using the novel method of cascade averaging of hydrophobicity function within the limits of a sliding window. The proposed method allowed revealing 139 transmembrane domains out of 140 (or 99.3%) identified by other methods. Also 236 transmembrane domain boundary positions out of 280 (or 84%) were predicted correctly by the proposed method with deviation from the predictions made by other methods that does not exceed the detection error of this method.

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Induction of Rare Conformation of Oligosaccharide by Binding to Calcium-dependent Bacterial Lectin: X-ray Crystallography and Modelling Study

10.1101/612689 ◽

2019 ◽

Author(s):

Martin Lepsik ◽

Roman Sommer ◽

Sakonwan Kuhaudomlarp ◽

Mickaёl Lelimousin ◽

Emanuele Paci ◽

...

Keyword(s):

Force Field ◽

Three Dimensional ◽

Dimensional Structure ◽

Lewis X ◽

X Ray ◽

X Ray Crystallography ◽

Calcium Dependent ◽

Protein Receptors ◽

Dependent Protein ◽

Micro Organisms

ABSTRACTPathogenic micro-organisms utilize protein receptors in adhesion to host tissues, a process that in some cases relies on the interaction between lectin and human glycoconjugates. Oligosaccharide epitopes are recognized through their three-dimensional structure and their flexibility is a key issue in specificity. In this paper, we analyse by X-ray crystallography the structures of the lectin LecB from two strains of Pseudomonas aeruginosa in complex with Lewis x oligosaccharide present on cell surfaces of human tissues. An unusual conformation of the glycan was observed in all binding sites with a non-canonical syn orientation of the N-acetyl group of N-acetyl-glucosamine. A PDB-wide search revealed that such an orientation occurs only in 2% of protein/carbohydrate complexes. Theoretical chemistry calculations showed that the observed conformation is unstable in solution but stabilised by the lectin. A reliable description of LecB/Lewis x complex by force field-based methods had proven as especially challenging due to the special feature of the binding site, two closely apposed Ca2+ ions which induce strong charge delocalisation. By comparing various force-field parametrisations, we design general protocols which will be useful in near future for designing carbohydrate-based ligands (glycodrugs) against other calcium-dependent protein receptors.

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Architecturally complex O-glycopeptidases are customized for mucin recognition and hydrolysis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019220118 ◽

2021 ◽

Vol 118 (10) ◽

pp. e2019220118

Author(s):

Benjamin Pluvinage ◽

Elizabeth Ficko-Blean ◽

Ilit Noach ◽

Christopher Stuart ◽

Nicole Thompson ◽

...

Keyword(s):

Three Dimensional ◽

Peptide Bond ◽

Molecular Organization ◽

Dimensional Structure ◽

Carbohydrate Binding ◽

Functional Roles ◽

Protein Substrates ◽

Individual Domain ◽

Insight Into

A challenge faced by peptidases is the recognition of highly diverse substrates. A feature of some peptidase families is the capacity to specifically use post-translationally added glycans present on their protein substrates as a recognition determinant. This is ultimately critical to enabling peptide bond hydrolysis. This class of enzyme is also frequently large and architecturally sophisticated. However, the molecular details underpinning glycan recognition by these O-glycopeptidases, the importance of these interactions, and the functional roles of their ancillary domains remain unclear. Here, using the Clostridium perfringens ZmpA, ZmpB, and ZmpC M60 peptidases as model proteins, we provide structural and functional insight into how these intricate proteins recognize glycans as part of catalytic and noncatalytic substrate recognition. Structural, kinetic, and mutagenic analyses support the key role of glycan recognition within the M60 domain catalytic site, though they point to ZmpA as an apparently inactive enzyme. Wider examination of the Zmp domain content reveals noncatalytic carbohydrate binding as a feature of these proteins. The complete three-dimensional structure of ZmpB provides rare insight into the overall molecular organization of a highly multimodular enzyme and reveals how the interplay of individual domain function may influence biological activity. O-glycopeptidases frequently occur in host-adapted microbes that inhabit or attack mucus layers. Therefore, we anticipate that these results will be fundamental to informing more detailed models of how the glycoproteins that are abundant in mucus are destroyed as part of pathogenic processes or liberated as energy sources during normal commensal lifestyles.

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