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Rhiju
Das
,
Rachael C.
Kretsch
,
Adam J.
Simpkin
,
Thomas
Mulvaney
,
Phillip
Pham
,
Ramya
Rangan
,
Fan
Bu
,
Ronan M.
Keegan
,
Maya
Topf
,
Daniel J.
Rigden
,
Zhichao
Miao
,
Eric
Westhof
Open Access
Abstract: The prediction of RNA three-dimensional structures remains an unsolved problem. Here, we report assessments of RNA structure predictions in CASP15, the first CASP exercise that involved RNA structure modeling. Forty-two predictor groups submitted models for at least one of twelve RNA-containing targets. These models were evaluated by the RNA-Puzzles organizers and, separately, by a CASP-recruited team using metrics (GDT, lDDT) and approaches (Z-score rankings) initially developed for assessment of proteins and generalized here for RNA assessment. The two assessments independently ranked the same predictor groups as first (AIchemy_RNA2), second (Chen), and third (RNAPolis and GeneSilico, tied); predictions from deep learning approaches were significantly worse than these top ranked groups, which did not use deep learning. Further analyses based on direct comparison of predicted models to cryogenic electron microscopy (cryo-EM) maps and x-ray diffraction data support these rankings. With the exception of two RNA-protein complexes, models submitted by CASP15 groups correctly predicted the global fold of the RNA targets. Comparisons of CASP15 submissions to designed RNA nanostructures as well as molecular replacement trials highlight the potential utility of current RNA modeling approaches for RNA nanotechnology and structural biology, respectively. Nevertheless, challenges remain in modeling fine details such as noncanonical pairs, in ranking among submitted models, and in prediction of multiple structures resolved by cryo-EM or crystallography.
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Oct 2023
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VMXm-Versatile Macromolecular Crystallography microfocus
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Leila T.
Alexander
,
Janani
Durairaj
,
Andriy
Kryshtafovych
,
Luciano A.
Abriata
,
Yusupha
Bayo
,
Gira
Bhabha
,
Cécile
Breyton
,
Simon G.
Caulton
,
James
Chen
,
Séraphine
Degroux
,
Damian C.
Ekiert
,
Benedikte S.
Erlandsen
,
Peter L.
Freddolino
,
Dominic
Gilzer
,
Chris
Greening
,
Jonathan M.
Grimes
,
Rhys
Grinter
,
Manickam
Gurusaran
,
Marcus D.
Hartmann
,
Charlie J.
Hitchman
,
Jeremy R.
Keown
,
Ashleigh
Kropp
,
Petri
Kursula
,
Andrew L.
Lovering
,
Bruno
Lemaitre
,
Andrea
Lia
,
Shiheng
Liu
,
Maria
Logotheti
,
Shuze
Lu
,
Sigurbjorn
Markusson
,
Mitchell D.
Miller
,
George
Minasov
,
Hartmut H.
Niemann
,
Felipe
Opazo
,
George N.
Phillips
,
Owen R.
Davies
,
Samuel
Rommelaere
,
Monica
Rosas‐lemus
,
Pietro
Roversi
,
Karla
Satchell
,
Nathan
Smith
,
Mark A.
Wilson
,
Kuan‐lin
Wu
,
Xian
Xia
,
Han
Xiao
,
Wenhua
Zhang
,
Z. Hong
Zhou
,
Krzysztof
Fidelis
,
Maya
Topf
,
John
Moult
,
Torsten
Schwede
Diamond Proposal Number(s):
[19946, 23570, 27314, 28534]
Open Access
Abstract: We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.
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Jul 2023
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Krios I-Titan Krios I at Diamond
|
Diamond Proposal Number(s):
[14704]
Open Access
Abstract: Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with proapoptotic granzymes, to bind to a target cell membrane where it oligomerizes and forms pores. The pores allow granzyme entry, which rapidly triggers the apoptotic death of the target cell. Here, we present a 4-Å resolution cryo–electron microscopy structure of the perforin pore, revealing previously unidentified inter- and intramolecular interactions stabilizing the assembly.
During pore formation, the helix-turn-helix motif moves away from the bend in the central beta-sheet to form an intermolecular contact. Cryo–electron tomography shows that prepores form on the membrane surface with minimal conformational changes. Our findings suggest the sequence of conformational changes underlying oligomerization and membrane insertion, and explain how several pathogenic mutations affect function.
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Feb 2022
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I03-Macromolecular Crystallography
|
Leila T.
Alexander
,
Rosalba
Lepore
,
Andriy
Kryshtafovych
,
Athanasios
Adamopoulos
,
Markus
Alahuhta
,
Ann M.
Arvin
,
Yannick J.
Bomble
,
Bettina
Böttcher
,
Cécile
Breyton
,
Valerio
Chiarini
,
Naga Babu
Chinnam
,
Wah
Chiu
,
Krzysztof
Fidelis
,
Rhys
Grinter
,
Gagan D.
Gupta
,
Marcus D.
Hartmann
,
Christopher S.
Hayes
,
Tatjana
Heidebrecht
,
Andrea
Ilari
,
Andrzej
Joachimiak
,
Youngchang
Kim
,
Romain
Linares
,
Andrew L.
Lovering
,
Vladimir V.
Lunin
,
Andrei N.
Lupas
,
Cihan
Makbul
,
Karolina
Michalska
,
John
Moult
,
Prasun K.
Mukherjee
,
William
Nutt
,
Stefan L.
Oliver
,
Anastassis
Perrakis
,
Lucy
Stols
,
John A.
Tainer
,
Maya
Topf
,
Susan E.
Tsutakawa
,
Mauricio
Valdivia‐delgado
,
Torsten
Schwede
Open Access
Abstract: The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.
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Dec 2021
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Krios I-Titan Krios I at Diamond
|
B.
Vollmer
,
V.
Prazak
,
D.
Vasishtan
,
E. E.
Jefferys
,
A.
Hernandez-Duran
,
M.
Vallbracht
,
B. G.
Klupp
,
T. C.
Mettenleiter
,
M.
Backovic
,
F. A.
Rey
,
M.
Topf
,
K.
Grunewald
Open Access
Abstract: Cell entry of enveloped viruses requires specialized viral proteins that mediate fusion with the host membrane by substantial structural rearrangements from a metastable pre- to a stable postfusion conformation. This metastability renders the herpes simplex virus 1 (HSV-1) fusion glycoprotein B (gB) highly unstable such that it readily converts into the postfusion form, thereby precluding structural elucidation of the pharmacologically relevant prefusion conformation. By identification of conserved sequence signatures and molecular dynamics simulations, we devised a mutation that stabilized this form. Functionally locking gB allowed the structural determination of its membrane-embedded prefusion conformation at sub-nanometer resolution and enabled the unambiguous fit of all ectodomains. The resulting pseudo-atomic model reveals a notable conservation of conformational domain rearrangements during fusion between HSV-1 gB and the vesicular stomatitis virus glycoprotein G, despite their very distant phylogeny. In combination with our comparative sequence-structure analysis, these findings suggest common fusogenic domain rearrangements in all class III viral fusion proteins.
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Sep 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Rosalba
Lepore
,
Andriy
Kryshtafovych
,
Markus
Alahuhta
,
Harshul A.
Veraszto
,
Yannick J.
Bomble
,
Joshua C.
Bufton
,
Alex N.
Bullock
,
Cody
Caba
,
Hongnan
Cao
,
Owen R.
Davies
,
Ambroise
Desfosses
,
Matthew
Dunne
,
Krzysztof
Fidelis
,
Celia W.
Goulding
,
Manickam
Gurusaran
,
Irina
Gutsche
,
Christopher J.
Harding
,
Marcus D.
Hartmann
,
Christopher S.
Hayes
,
Andrzej
Joachimiak
,
Petr G.
Leiman
,
Peter
Loppnau
,
Andrew L.
Lovering
,
Vladimir V.
Lunin
,
Karolina
Michalska
,
Ignacio
Mir‐sanchis
,
Alok
Mitra
,
John
Moult
,
George N.
Phillips Jr
,
Daniel
Pinkas
,
Phoebe A.
Rice
,
Yufeng
Tong
,
Maya
Topf
,
Jonathan D.
Walton
,
Torsten
Schwede
Diamond Proposal Number(s):
[14692]
Open Access
Abstract: The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.
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Sep 2019
|
|
Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
|
Diamond Proposal Number(s):
[19429, 13304, 16919, 13893, 12204, 12388]
Open Access
Abstract: The membrane attack complex (MAC) is one of the immune system’s first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant β-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how β-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.
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Dec 2018
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|
Krios I-Titan Krios I at Diamond
|
Diamond Proposal Number(s):
[14704]
Open Access
Abstract: Refolding aggregated proteins is essential in combating cellular proteotoxic stress. Together with Hsp70, Hsp100 chaperones, including Escherichia coli ClpB, form a powerful disaggregation machine that threads aggregated polypeptides through the central pore of tandem adenosine triphosphatase (ATPase) rings. To visualize protein disaggregation, we determined cryo–electron microscopy structures of inactive and substrate-bound ClpB in the presence of adenosine 5′-O-(3-thiotriphosphate), revealing closed AAA+ rings with a pronounced seam. In the substrate-free state, a marked gradient of resolution, likely corresponding to mobility, spans across the AAA+ rings with a dynamic hotspot at the seam. On the seam side, the coiled-coil regulatory domains are locked in a horizontal, inactive orientation. On the opposite side, the regulatory domains are accessible for Hsp70 binding, substrate targeting, and activation. In the presence of the model substrate casein, the polypeptide threads through the entire pore channel and increased nucleotide occupancy correlates with higher ATPase activity. Substrate-induced domain displacements indicate a pathway of regulated substrate transfer from Hsp70 to the ClpB pore, inside which a spiral of loops contacts the substrate. The seam pore loops undergo marked displacements, along with ordering of the regulatory domains. These asymmetric movements suggest a mechanism for ATPase activation and substrate threading during disaggregation.
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Aug 2017
|
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Krios I-Titan Krios I at Diamond
|
Open Access
Abstract: As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high resolutions and robust approaches for model assessment. Flex-EM/MODELLER has been used for flexible fitting of atomic models in intermediate-to-low resolution density maps of different biological systems. Here, we demonstrate the suitability of the method to successfully refine structures at higher resolutions (2.5-4.5 Å) using both simulated and experimental data, including a newly processed map of Apo-GroEL. A hierarchical refinement protocol was adopted where the rigid body definitions are relaxed and atom displacement steps are reduced progressively at successive stages of refinement. For the assessment of local fit, we used the SMOC (segment-based Manders’ overlap coefficient) score, while the model quality was checked using the Qmean score. Comparison of SMOC profiles at different stages of refinement helped in detecting regions that are poorly fitted. We also show how initial model errors can have significant impact on the goodness-of-fit. Finally, we discuss the implementation of Flex-EM in the CCP-EM software suite.
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Mar 2016
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