I24-Microfocus Macromolecular Crystallography
|
Agata
Butryn
,
Philipp S.
Simon
,
Pierre
Aller
,
Philip
Hinchliffe
,
Ramzi N.
Massad
,
Gabriel
Leen
,
Catherine L.
Tooke
,
Isabel
Bogacz
,
In-Sik
Kim
,
Asmit
Bhowmick
,
Aaron S.
Brewster
,
Nicholas E.
Devenish
,
Jurgen
Brem
,
Jos J. A. G.
Kamps
,
Pauline A.
Lang
,
Patrick
Rabe
,
Danny
Axford
,
John H.
Beale
,
Bradley
Davy
,
Ali
Ebrahim
,
Julien
Orlans
,
Selina L. S.
Storm
,
Tiankun
Zhou
,
Shigeki
Owada
,
Rie
Tanaka
,
Kensuke
Tono
,
Gwyndaf
Evans
,
Robin L.
Owen
,
Frances A.
Houle
,
Nicholas K.
Sauter
,
Christopher J.
Schofield
,
James
Spencer
,
Vittal K.
Yachandra
,
Junko
Yano
,
Jan F.
Kern
,
Allen M.
Orville
Diamond Proposal Number(s):
[19458, 25260]
Open Access
Abstract: Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.
|
Jul 2021
|
|
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Julien
Orlans
,
Carole
Vincent-Monegat
,
Isabelle
Rahioui
,
Catherine
Sivignon
,
Agata
Butryn
,
Laurent
Soulère
,
Anna
Zaidman-Remy
,
Allen M.
Orville
,
Abdelaziz
Heddi
,
Pierre
Aller
,
Pedro
Da Silva
Diamond Proposal Number(s):
[19737, 19458, 23571]
Open Access
Abstract: Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso-2,6-diaminopimelic (meso-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn2+.
|
May 2021
|
|
|
|
Abstract: Macromolecular crystallography (MX) leverages the methods of physics and the language of chemistry to reveal fundamental insights into biology. Often beautifully artistic images present MX results to support profound functional hypotheses that are vital to entire life science research community. Over the past several decades, synchrotrons around the world have been the workhorses for X-ray diffraction data collection at many highly automated beamlines. The newest tools include X-ray-free electron lasers (XFELs) located at facilities in the USA, Japan, Korea, Switzerland, and Germany that deliver about nine orders of magnitude higher brightness in discrete femtosecond long pulses. At each of these facilities, new serial femtosecond crystallography (SFX) strategies exploit slurries of micron-size crystals by rapidly delivering individual crystals into the XFEL X-ray interaction region, from which one diffraction pattern is collected per crystal before it is destroyed by the intense X-ray pulse. Relatively simple adaptions to SFX methods produce time-resolved data collection strategies wherein reactions are triggered by visible light illumination or by chemical diffusion/mixing. Thus, XFELs provide new opportunities for high temporal and spatial resolution studies of systems engaged in function at physiological temperature. In this chapter, we summarize various issues related to microcrystal slurry preparation, sample delivery into the X-ray interaction region, and some emerging strategies for time-resolved SFX data collection.
|
May 2021
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Anka
Lucic
,
Philip
Hinchliffe
,
Tika R.
Malla
,
Catherine L.
Tooke
,
Jurgen
Brem
,
Karina
Calvopina
,
Christopher T.
Lohans
,
Patrick
Rabe
,
Michael A.
Mcdonough
,
Timothy
Armistead
,
Allen M.
Orville
,
James
Spencer
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[17212, 23269, 18069]
Abstract: Penems have demonstrated potential as antibacterials and β-lactamase inhibitors; however, their clinical use has been limited, especially in comparison with the structurally related carbapenems. Faropenem is an orally active antibiotic with a C2 tetrahydrofuran (THF) ring, which is resistant to hydrolysis by some β-lactamases. We report studies on the reactions of faropenem with carbapenem-hydrolysing β-lactamases, focusing on the class A serine β-lactamase KPC-2 and the metallo β-lactamases (MBLs) VIM-2 (a subclass B1 MBL) and L1 (a B3 MBL). Kinetic studies show that faropenem is a substrate for all three β-lactamases, though it is less efficiently hydrolysed by KPC-2. Crystallographic analyses on faropenem-derived complexes reveal the opening of the β-lactam ring with formation of an imine with KPC-2, VIM-2, and L1. In the cases of the KPC-2 and VIM-2 structures, the THF ring is opened to give an alkene, but with L1 the THF ring remains intact. Solution state studies, employing NMR, were performed on L1, KPC-2, VIM-2, VIM-1, NDM-1, OXA-23, OXA-10, and OXA-48. The solution results reveal, in all cases, formation of imine products in which the THF ring is opened; formation of a THF ring-closed imine product was only observed with VIM-1 and VIM-2. An enamine product with a closed THF ring was also observed in all cases, at varying levels. Combined with previous reports, the results exemplify the potential for different outcomes in the reactions of penems with MBLs and SBLs and imply further structure-activity relationship studies are worthwhile to optimise the interactions of penems with β-lactamases. They also exemplify how crystal structures of β-lactamase substrate/inhibitor complexes do not always reflect reaction outcomes in solution.
|
Feb 2021
|
|
|
|
Open Access
Abstract: Time-resolved serial femtosecond crystallography (tr-SFX) methods exploit slurries of crystalline samples that range in size from hundreds of nanometers to a few tens of micrometers, at near-physiological temperature and pressure, to generate atomic resolution models and probe authentic function with the same experiment. ‘Dynamic structural biology’ is often used to encompass the research philosophy and techniques. Reaction cycles for tr-SFX studies are initiated by photons or ligand addition/mixing strategies, wherein the latter are potentially generalizable across enzymology. Thus, dynamic structural biology often creates stop-motion molecular movies of macromolecular function. In metal-dependent systems, complementary spectroscopic information can also be collected from the same samples and X-ray pulses, which provides even more detailed mechanistic insights. These types of experimental data also complement quantum mechanical and classical dynamics numerical calculations. Correlated structural-functional results will yield more detailed mechanistic insights and will likely translate into better drugs and treatments impacting human health, and better catalysis for clean energy and agriculture.
|
Dec 2020
|
|
I24-Microfocus Macromolecular Crystallography
|
Patrick
Rabe
,
John
Beale
,
Agata
Butryn
,
Pierre
Aller
,
Anna
Dirr
,
Pauline A.
Lang
,
Danny N.
Axford
,
Stephen
Carr
,
Thomas M.
Leissing
,
Michael A.
Mcdonough
,
Bradley
Davy
,
Ali
Ebrahim
,
Julien
Orlans
,
Selina L. S.
Storm
,
Allen M.
Orville
,
Christopher J.
Schofield
,
Robin L.
Owen
Diamond Proposal Number(s):
[19458]
Open Access
Abstract: Cryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryo-conditions (usually employing a nitrogen cryo-stream at 100 K) enable, is data collection of dioxygen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O2 diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for dioxygen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the `sheet-on-sheet' sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (<5 min). The utility of the method is highlighted by studies with three X-ray-radiation-sensitive Fe(II)-containing model enzymes: the 2-oxoglutarate-dependent L-arginine hydroxylase VioC and the DNA repair enzyme AlkB, as well as the oxidase isopenicillin N synthase (IPNS), which is involved in the biosynthesis of all penicillin and cephalosporin antibiotics.
|
Sep 2020
|
|
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[14449, 16818]
Open Access
Abstract: In this article, a new approach to experimental phasing for macromolecular crystallography (MX) at synchrotrons is introduced and described for the first time. It makes use of automated robotics applied to a multi-crystal framework in which human intervention is reduced to a minimum. Hundreds of samples are automatically soaked in heavy-atom solutions, using a Labcyte Inc. Echo 550 Liquid Handler, in a highly controlled and optimized fashion in order to generate derivatized and isomorphous crystals. Partial data sets obtained on MX beamlines using an in situ setup for data collection are processed with the aim of producing good-quality anomalous signal leading to successful experimental phasing.
|
Aug 2020
|
|
|
|
Vivek
Srinivas
,
Rahul
Banerjee
,
Hugo
Lebrette
,
Jason C.
Jones
,
Oskar
Aurelius
,
In-Sik
Kim
,
Cindy C.
Pham
,
Sheraz
Gul
,
Kyle
Sutherlin
,
Asmit
Bhowmick
,
Juliane
John
,
Esra
Bozkurt
,
Thomas
Fransson
,
Pierre
Aller
,
Agata
Butryn
,
Isabel
Bogacz
,
Philipp Stefan
Simon
,
Stephen
Keable
,
Alexander
Britz
,
Kensuke
Tono
,
Kyung-Sook
Kim
,
Sang-Youn
Park
,
Sang-Jae
Lee
,
Jaehyun
Park
,
Roberto
Alonso-Mori
,
Franklin
Fuller
,
Alexander
Batyuk
,
Aaron S.
Brewster
,
Uwe
Bergmann
,
Nicholas
Sauter
,
Allen M.
Orville
,
Vittal K.
Yachandra
,
Junko
Yano
,
John D.
Lipscomb
,
Jan F.
Kern
,
Martin
Högbom
Abstract: Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O2 activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of pure oxidation states. Here microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≦35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.
|
Jul 2020
|
|
|
|
Mohamed
Ibrahim
,
Thomas
Fransson
,
Ruchira
Chatterjee
,
Mun Hon
Cheah
,
Rana
Hussein
,
Louise
Lassalle
,
Kyle D.
Sutherlin
,
Iris D.
Young
,
Franklin D.
Fuller
,
Sheraz
Gul
,
In-Sik
Kim
,
Philipp S.
Simon
,
Casper
De Lichtenberg
,
Petko
Chernev
,
Isabel
Bogacz
,
Cindy C.
Pham
,
Allen M.
Orville
,
Nicholas
Saichek
,
Trent
Northen
,
Alexander
Batyuk
,
Sergio
Carbajo
,
Roberto
Alonso-Mori
,
Kensuke
Tono
,
Shigeki
Owada
,
Asmit
Bhowmick
,
Robert
Bolotovsky
,
Derek
Mendez
,
Nigel W.
Moriarty
,
James M.
Holton
,
Holger
Dobbek
,
Aaron S.
Brewster
,
Paul D.
Adams
,
Nicholas K.
Sauter
,
Uwe
Bergmann
,
Athina
Zouni
,
Johannes
Messinger
,
Jan
Kern
,
Vittal K.
Yachandra
,
Junko
Yano
Open Access
Abstract: In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a “water wheel”-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2 → S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.
|
May 2020
|
|
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
|
Diamond Proposal Number(s):
[16818, 19737, 19485]
Open Access
Abstract: Cyclic guanosine 3′,5′‐monophosphate (cGMP) is an intracellular signaling molecule involved in many sensory and developmental processes. Synthesis of cGMP from GTP is catalyzed by guanylate cyclase (GC) in a reaction analogous to cAMP formation by adenylate cyclase (AC). Although detailed structural information is available on the catalytic region of nucleotidyl cyclases (NCs) in various states, these atomic models do not provide a sufficient explanation for the substrate selectivity between GC and AC family members. Detailed structural information on the GC domain in its active conformation is largely missing and no crystal structure of a GTP‐bound wild‐type GC domain has been published to date. Here, we describe the crystal structure of the catalytic domain of rhodopsin‐GC (RhGC) from Catenaria anguillulae in complex with GTP at 1.7 Å resolution. Our study reveals the organization of a eukaryotic GC domain in its active conformation. We observe that the binding mode of the substrate GTP is similar to that of AC–ATP interaction, although surprisingly not all of the interactions predicted to be responsible for base recognition are present. The structure provides insights into potential mechanisms of substrate discrimination and activity regulation that may be common to all class III purine NCs.
|
Dec 2019
|
|
