I24-Microfocus Macromolecular Crystallography
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Simon
D'Oelsnitz
,
Wantae
Kim
,
Nicole N.
Zhao
,
Haley
Hardtke
,
Svetlana P.
Ikonomova
,
Nina
Alperovich
,
Olga
Vasilyeva
,
Michael J.
James
,
Eric S.
Zigon
,
Michael B.
Cory
,
Charlie D.
Johnson
,
Andrew D.
Ellington
,
Quincey A.
Justman
,
Michael
Springer
,
Y. Jessie
Zhang
,
Pamela A.
Silver
,
David
Ross
Diamond Proposal Number(s):
[36008]
Open Access
Abstract: Biocatalysts are prized for their enantioselectivity, but slow chromatographic separations required to measure enantiomeric excess bottleneck their development. To overcome this limitation, we evolve enantioselective transcription factors (eTFs) that convert enzyme-catalyzed enantiomer concentrations into programmable gene expression outputs, focusing on imine reductases. Here, using a massively parallel reporter assay, we measure dose–response curves for over 300,000 transcription factor variants in response to an imine precursor and chiral amine products. We quantify the sensitivity, selectivity and dynamic range across variants generated by random, site-saturation and shuffling mutagenesis, isolating variants with exceptional specificity. High-resolution structures of evolved eTFs elucidate how steric effects enforce enantioselectivity, while charge interactions distinguish the imine from the amines. Using two eTFs, we create an ultrahigh-throughput chiral screen to evolve an imine reductase with inverted enantioselectivity. To support generalizability and speed, we design a genetic circuit that enables TF generation within weeks. Our methods enable rapid measurement of asymmetric reactions, supporting innovation in chemical manufacturing.
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Jul 2026
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Krios II-Titan Krios II at Diamond
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Valentina A.
Spiteri
,
Dmitri
Segal
,
Alejandro
Correa-Sáez
,
Kentaro
Iso
,
Ryan
Casement
,
Miquel
Muñoz I Ordoño
,
Mark A.
Nakasone
,
Gajanan
Sathe
,
Caroline
Schätz
,
Hannah E.
Peters
,
Mark
Doward
,
Lisa
Kainacher
,
Angus D.
Cowan
,
Alessio
Ciulli
,
Georg E.
Winter
Diamond Proposal Number(s):
[37630]
Open Access
Abstract: Proteolysis-targeting chimeras (PROTACs) and molecular glue degraders (MGDs) target proteins for degradation by co-opting an E3 ligase. While heterotrivalent PROTACs that can recruit multiple E3 ligases have been described, all MGDs reported to date depend on a single E3. Using orthogonal genetic screening, biophysical and structural analyses, we show that a monovalent MGD can recruit CUL4DCAF16 and CRL1FBXO22 in parallel to degrade SMARCA2/4. Deep mutational scanning identifies C173 in DCAF16 as essential for degrader activity and intact protein mass spectrometry confirms covalent modification at this site. Elucidating the ternary complex structure reveals a unique binding mode and a distinct interface of neointeractions that underlie degrader specificity. We demonstrate that ligase dependency is chemically and genetically tunable. Minimal compound modifications shift preference from DCAF16 to FBXO22, while a single substitution boosts degrader dependency on DCAF16. These results establish a framework for designing tunable dual E3 ligase degraders to mitigate potential resistance mechanisms.
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May 2026
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I24-Microfocus Macromolecular Crystallography
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James B.
Shaum
,
Miquel
Muñoz I Ordoño
,
Erica A.
Steen
,
Daniela V.
Wenge
,
Hakyung
Cheong
,
Jordan
Janowski
,
Moritz
Hunkeler
,
Eric M.
Bilotta
,
Zoe J.
Rutter
,
Paige A.
Barta
,
Abby M.
Thornhill
,
Natalia
Milosevich
,
Lauren M.
Hargis
,
Timothy R.
Bishop
,
Trever R.
Carter
,
Bryce
Da Camara
,
Matthias
Hinterndorfer
,
Lucas
Dada
,
Wen-Ji
He
,
Fabian
Offensperger
,
Hirotake
Furihata
,
Sydney R.
Schweber
,
Charlie
Hatton
,
Yanhe
Wen
,
Benjamin F.
Cravatt
,
Keary M.
Engle
,
Katherine A.
Donovan
,
Bruno
Melillo
,
Seiya
Kitamura
,
Alessio
Ciulli
,
Scott A.
Armstrong
,
Eric S.
Fischer
,
Georg E.
Winter
,
Michael A.
Erb
Diamond Proposal Number(s):
[35324]
Open Access
Abstract: Chemical inducers of proximity (CIPs) stabilize biomolecular interactions, often causing an emergent rewiring of cellular biochemistry. While the discovery of heterobifunctional CIPs is expedited by rational design strategies, molecular glues have relied predominantly on serendipity. We hypothesized that preexisting ligands could be systematically decorated with chemical modifications to discover compounds that recruit proteins to a composite protein–ligand interface. Using sulfur(VI) fluoride exchange-based high-throughput chemistry (HTC) to install 3,163 structurally diverse building blocks onto ENL (eleven-nineteen leukemia) and BRD4 (bromodomain-containing protein 4) ligands, we screened each analog for degrader activity. This revealed dHTC1, an ENL degrader that recruits CRL4CRBN complex through an extended interface of protein–protein contacts and only engages CRBN after pre-forming the ENL:dHTC1 complex. We also identified dHTC3, a molecular glue that selectively dimerizes BRD4 bromodomain 1 to SCFFBXO3, an E3 ligase not previously accessible for chemical rewiring. Altogether, this study introduces HTC as a facile tool to discover new CIPs and new effectors for proximity pharmacology.
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Feb 2026
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Krios I-Titan Krios I at Diamond
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Gangshun
Yi
,
Dimitrios
Mamalis
,
Mingda
Ye
,
Loic
Carrique
,
Michael
Fairhead
,
Huanyu
Li
,
Katharina L.
Duerr
,
Peijun
Zhang
,
David B.
Sauer
,
Frank
Von Delft
,
Benjamin G.
Davis
,
Robert J. C.
Gilbert
Diamond Proposal Number(s):
[20223, 21004]
Open Access
Abstract: Whilst cryo-electron microscopy(cryo-EM) has become a routine methodology in structural biology, obtaining high-resolution cryo-EM structures of small proteins (<100 kDa) and increasing overall throughput remain challenging. One approach to augment protein size and improve particle alignment involves the use of binding proteins or protein-based scaffolds. However, a given imaging scaffold or linking module may prove inadequate for structure solution and availability of such scaffolds remains limited. Here, we describe a strategy that exploits covalent dimerization of nanobodies to trap an engineered, predisposed nanobody-to-nanobody interface, giving Di-Gembodies as modular constructs created in homomeric and heteromeric forms. By exploiting side-chain-to-side-chain assembly, they can simultaneously display two copies of the same or two distinct proteins through a subunit interface that provides sufficient constraint required for cryo-EM structure determination. We validate this method with multiple soluble and membrane structural targets, down to 14 kDa, demonstrating a flexible and scalable platform for expanded protein structure determination.
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Aug 2025
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I24-Microfocus Macromolecular Crystallography
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Ramon
Hurtado-Guerrero
,
Spyridon
Gatos
,
Irene
Ginés-Alcober
,
Javier
Macías-León
,
Andrés Manuel
González-Ramírez
,
Ilknur
Kasapoglu
,
Billy
Veloz
,
Ismael
Compañón
,
Mattia
Ghirardello
,
Pedro
Merino
,
Francisco
Corzana
,
Ola
Blixt
Diamond Proposal Number(s):
[20229]
Abstract: Developing high-affinity monoclonal antibodies (mAbs) against tumor-associated carbohydrate antigens such as Tn and STn on carrier proteins remains a major challenge in cancer therapy. These antigens, expressed as glycan–peptide epitopes (combotopes), require precise recognition for high specificity. Through structural studies, we found that VH domains of certain antibodies primarily recognize glycans, whereas VL domains bind peptide sequences. Using these insights, we developed a VH-focused and VL-diverse phage display library to discover mAbs with combotope-binding characteristics. Notably, structural analysis enabled us to convert Tn-specific mAbs into STn-specific mAbs through modification of VH complementarity-determining region 3, demonstrating the versatility of this approach. Our hypothesis was validated with glycoprotein targets MUC1 and CD43, yielding antibodies with high specificity and affinity. Furthermore, internalization studies using the parental antibody scaffold show efficient uptake by tumor cells, supporting its use in antibody–drug conjugates. This platform addresses the challenge of generating glycoform-specific antibodies for cancer therapy.
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Jul 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[40457, 32977, 32479]
Open Access
Abstract: The basic helix–loop–helix PER-ARNT-SIM (bHLH-PAS) proteins BMAL1 and CLOCK heterodimerize to form the master transcription factor governing rhythmic gene expression. Owing to connections between circadian regulation and numerous physiological pathways, targeting the BMAL1–CLOCK complex pharmacologically is an attractive entry point for intervening in circadian-related processes. In this study, we developed a small molecule, Core Circadian Modulator (CCM), that targets the cavity in the PASB domain of BMAL1, causing it to expand, leading to conformational changes in the PASB domain and altering the functions of BMAL1 as a transcription factor. Biochemical, structural and cellular investigations validate the high level of selectivity of CCM in engaging BMAL1, enabling direct access to BMAL1–CLOCK cellular activities. CCM induces dose-dependent alterations in PER2–Luc oscillations and orchestrates the downregulation of inflammatory and phagocytic pathways in macrophages. These findings collectively reveal that the BMAL1 protein architecture is inherently configured to enable the binding of chemical ligands for functional modulation.
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Mar 2025
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I24-Microfocus Macromolecular Crystallography
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Christa
Litschko
,
Valerio
Di Domenico
,
Julia
Schultz
,
Sizhe
Li
,
Olga G.
Ovchinnikova
,
Thijs
Voskuilen
,
Andrea
Bethe
,
Javier O.
Cifuente
,
Alberto
Marina
,
Insa
Budde
,
Tim A.
Mast
,
Małgorzata
Sulewska
,
Monika
Berger
,
Falk F. R.
Buettner
,
Todd L.
Lowary
,
Chris
Whitfield
,
Jeroen D. C.
Codée
,
Mario
Schubert
,
Marcelo E.
Guerin
,
Timm
Fiebig
Diamond Proposal Number(s):
[28360]
Open Access
Abstract: Capsules are long-chain carbohydrate polymers that envelop the surfaces of many bacteria, protecting them from host immune responses. Capsule biosynthesis enzymes are potential drug targets and valuable biotechnological tools for generating vaccine antigens. Despite their importance, it remains unknown how structurally variable capsule polymers of Gram-negative pathogens are linked to the conserved glycolipid anchoring these virulence factors to the bacterial membrane. Using Actinobacillus pleuropneumoniae as an example, we demonstrate that CpsA and CpsC generate a poly(glycerol-3-phosphate) linker to connect the glycolipid with capsules containing poly(galactosylglycerol-phosphate) backbones. We reconstruct the entire capsule biosynthesis pathway in A. pleuropneumoniae serotypes 3 and 7, solve the X-ray crystal structure of the capsule polymerase CpsD, identify its tetratricopeptide repeat domain as essential for elongating poly(glycerol-3-phosphate) and show that CpsA and CpsC stimulate CpsD to produce longer polymers. We identify the CpsA and CpsC product as a wall teichoic acid homolog, demonstrating similarity between the biosynthesis of Gram-positive wall teichoic acid and Gram-negative capsules.
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Jul 2024
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I04-Macromolecular Crystallography
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Guangcai
Xu
,
Daniele
Torri
,
Sebastian
Cuesta-Hoyos
,
Deepanjan
Panda
,
Luke R. L.
Yates
,
Rémi
Zallot
,
Kehan
Bian
,
Dongxu
Jia
,
Andreea I.
Iorgu
,
Colin
Levy
,
Sarah A.
Shepherd
,
Jason
Micklefield
Diamond Proposal Number(s):
[31850]
Open Access
Abstract: Nature has evolved biosynthetic pathways to molecules possessing reactive warheads that inspired the development of many therapeutic agents, including penicillin antibiotics. Peptides armed with electrophilic warheads have proven to be particularly effective covalent inhibitors, providing essential antimicrobial, antiviral and anticancer agents. Here we provide a full characterization of the pathways that nature deploys to assemble peptides with β-lactone warheads, which are potent proteasome inhibitors with promising anticancer activity. Warhead assembly involves a three-step cryptic methylation sequence, which is likely required to reduce unfavorable electrostatic interactions during the sterically demanding β-lactonization. Amide-bond synthetase and adenosine triphosphate (ATP)-grasp enzymes couple amino acids to the β-lactone warhead, generating the bioactive peptide products. After reconstituting the entire pathway to β-lactone peptides in vitro, we go on to deliver a diverse range of analogs through enzymatic cascade reactions. Our approach is more efficient and cleaner than the synthetic methods currently used to produce clinically important warhead-containing peptides.
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Jul 2024
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[37518, 35672, 31440]
Open Access
Abstract: Many enzymes are allosterically regulated via conformational change; however, our ability to manipulate these structural changes and control function is limited. Here we install a conformational switch for allosteric activation into the kinesin-1 microtubule motor in vitro and in cells. Kinesin-1 is a heterotetramer that accesses open active and closed autoinhibited states. The equilibrium between these states centers on a flexible elbow within a complex coiled-coil architecture. We target the elbow to engineer a closed state that can be opened with a de novo designed peptide. The alternative states are modeled computationally and confirmed by biophysical measurements and electron microscopy. In cells, peptide-driven activation increases kinesin transport, demonstrating a primary role for conformational switching in regulating motor activity. The designs are enabled by our understanding of ubiquitous coiled-coil structures, opening possibilities for controlling other protein activities.
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Jun 2024
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Katherine I.
Albanese
,
Rokas
Petrenas
,
Fabio
Pirro
,
Elise A.
Naudin
,
Ufuk
Borucu
,
William M.
Dawson
,
D. Arne
Scott
,
Graham. J.
Leggett
,
Orion D.
Weiner
,
Thomas A. A.
Oliver
,
Derek N.
Woolfson
Diamond Proposal Number(s):
[23269, 31440]
Open Access
Abstract: Computational protein design is advancing rapidly. Here we describe efficient routes starting from validated parallel and antiparallel peptide assemblies to design two families of α-helical barrel proteins with central channels that bind small molecules. Computational designs are seeded by the sequences and structures of defined de novo oligomeric barrel-forming peptides, and adjacent helices are connected by loop building. For targets with antiparallel helices, short loops are sufficient. However, targets with parallel helices require longer connectors; namely, an outer layer of helix–turn–helix–turn–helix motifs that are packed onto the barrels. Throughout these computational pipelines, residues that define open states of the barrels are maintained. This minimizes sequence sampling, accelerating the design process. For each of six targets, just two to six synthetic genes are made for expression in Escherichia coli. On average, 70% of these genes express to give soluble monomeric proteins that are fully characterized, including high-resolution structures for most targets that match the design models with high accuracy.
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Jun 2024
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