Chapter Four · failure evidence
What X-Ray Diffraction & Crystallography got wrong, from 62 dissertations
The records document widespread experimental difficulties encountered across macromolecular crystallography, small molecule structure determination, and powder diffraction workflows. Researchers repeatedly faced barriers including failure to grow single crystals, crystal degradation during preparation, severe peak overlap during refinement, and resolution limits from weak scattering contrast. These records come from PhD theses at 18 institutions, 2021 to 2026. Each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Conformational heterogeneity and intrinsic disorder prevent the formation of diffraction quality macromolecular crystals
Protein constructs containing flexible termini, disordered loops, or conformational heterogeneity failed to yield usable crystals across extensive screening trials. When crystals did form, they diffracted weakly and to low resolution, preventing space group assignment or complete structure determination.
Tried and failed
X-ray crystallography applied to engineered protein loop deletion mutants. Outcome: no signal. Reason: Crystals could not be obtained or diffraction data failed to resolve structure
Elucidating novel chemical and genetic mechanisms of LSD1-HDAC1/2-CoREST complex regulation · Harvard
Tried and failed
X-ray crystallography of large macromolecular complexes applied to full-length protein-RNA initiation complex. Outcome: no signal. Reason: Crystals failed to produce measurable X-ray diffraction
Structural Understanding of Remodeling Function of HIV-1 Reverse Transcriptase During Initiation · Harvard
Tried and failed
protein-ligand cocrystallization for structure determination applied to enzyme-inhibitor complex. Outcome: no signal. Reason: crystals failed to form or yield ligand-bound diffraction data across multiple conditions
Structure-Based Discovery of Lipoteichoic Acid Synthase Inhibitors. · Cambridge
Tried and failed
X-ray crystallography applied to destabilized protein variants. Reason: High conformational heterogeneity and structural variability prevented crystal formation
Molecular Details of Amyloid Formation by the Glaucoma-Associated Myocilin Olfactomedin (OLF) Domain · Georgia Tech
Tried and failed
X-ray crystallography on heterooligomeric protein complexes applied to recombinant collagen C-propeptide heterotrimers. Outcome: no signal. Reason: crystals diffracted weakly and to low resolution, preventing space group assignment and structure determination
Structural and biochemical investigations of collagen-I trimerization · MIT
Tried and failed
crystal optimization and microseed matrix screening applied to recombinant bacterial protein crystallization. Outcome: no signal. Reason: failed to yield single, well-diffracting crystals suitable for X-ray diffraction
Tried and failed
fragment co-crystallization and soaking for X-ray crystallography applied to methyltransferase protein-ligand complex structure determination. Outcome: no signal. Reason: fragment screening hits failed to yield co-crystal structures via soaking or co-crystallization
Tried and failed
x-ray crystallography of full-length protein applied to protein with flexible c-terminal tail. Reason: the flexible c-terminus prevented crystal formation
The mechanism of Dnase1L3 mediated antigenic DNA clearance · Texas Tech
Tried and failed
X-ray crystallization of multi-protein complex applied to heteropentameric protein kinase complex. Outcome: no signal. Reason: Extensive crystallization screening failed to produce diffraction-quality crystals of the co-complex.
Structural and functional studies of the AMP-activated protein kinase γ2 isoform · Imperial
Considered and rejected
Considered and rejected: Rejected X-ray crystallography after repeatedly obtaining weakly diffracting (7.8–25 Å) crystals in favor of single-particle cryo-EM.
Structure and Function of the Alpha3Beta4 Nicotinic Acetylcholine Receptor · DSpace at UTSWMED
Considered and rejected
Considered and rejected: Abandoned X-ray crystallography optimization after initial 23Å diffraction in favor of single-particle cryo-EM due to poor protein yields and faster iteration cycles.
Considered and rejected
Considered and rejected: X-ray crystallography of 16 truncated APOLLO fragments was rejected after failing to induce most fragments and recognizing intrinsic disorder precludes accurate reconstituted structure determination
Structural analysis of APOLLO, a candidate apomixis factor · HARVEST
Considered and rejected
Considered and rejected: X-ray crystallography of CD163 SRCR1-5 construct was abandoned after cryo-EM revealed heterogeneous oligomerisation (dimers/trimers) and domain flexibility
Structural basis of haptoglobin-haemoglobin scavenging by CD163 · Oxford
Considered and rejected
Considered and rejected: Rejected co-crystallizing 17β-HSD14 at cold room conditions (~4°C) due to failure to form crystals.
Mutational Studies on 17β-HSD14, Serial Synchrotron X-ray Crystallography, Solubility Enhancement using Cyclodextrins and Fragment-Based Drug Discovery Multiple Blocks to Pave the Road of Drug Design · open_UMR Marburg DSpace 10.0
Considered and rejected
Considered and rejected: CRD-Fc fusion construct for X-ray crystallography (abandoned due to oligomeric heterogeneity and aberrant disulfide crosslinking).
Functional assessment of the cell entry pathway of Lymphocytic choriomeningitis virus · Harvard
Instrument resolution limits and low scattering contrast obscure dilute phases and light elements
Nanoscale phase dimensions, matrix dilution, and low analyte concentration caused diffraction signals to fall below instrument detection thresholds. In addition, weak scattering from light elements was heavily obscured by background noise or dominated by strong scattering from adjacent heavy atoms.
Tried and failed
X-ray diffraction crystal structure characterization applied to early-stage biopolymer development. Outcome: no signal. Reason: Molecular disorder and non-target matrix components obscure typical crystalline diffraction patterns.
Tried and failed
powder X-ray diffraction phase identification applied to surface-deposited metal oxide coatings on carbon. Outcome: no signal. Reason: Deposited oxides were amorphous, nanoscale, and present at low surface concentrations below detection thresholds.
Treatment of Aqueous Arsenic Using Chemically and Electrochemically Modified Biomaterials · HARVEST
Tried and failed
laboratory X-ray diffraction phase characterization applied to nanoscale secondary precipitates in metallic alloys. Outcome: no signal. Reason: Instrument resolution and signal overlap failed to resolve and differentiate nanoscale phase peaks from matrix phases
Microstructural Prediction of Additively Manufactured Multi-Phase Materials · Georgia Tech
Tried and failed
standard powder X-ray diffraction applied to micrometer-scale mineral structures. Reason: structures were too small (1 to 6 µm) to sample or analyze via standard powder XRD
Chemistry of the pearl oyster Pinctada radiata and implications for palaeoceanography · Queens University Institutional Repository
Tried and failed
generative structure prediction from powder diffraction patterns applied to crystals containing light elements. Outcome: no signal. Reason: Weak X-ray scattering of low-Z elements yields insufficient diffraction signal to resolve complex bonding structures.
Order Under Pressure: Structural and Magnetic Characterization at Extreme Stresses · MIT
Considered and rejected
Considered and rejected: Rejected relying on X-ray diffraction for high X-ray absorption samples (heavy fluxes like Bi or CsI), requiring neutron diffraction instead
Predictive Synthesis · Iowa State
Considered and rejected
Considered and rejected: Rejected relying on single-crystal XRD alone for structural validation of LiNiB and ternary phosphides because heavy transition metals (Ni, Ru, Ir) dominate scattering over light elements (Li, B, Si, P).
Improving sensitivity and resolution in solid-state nuclear magnetic resonance · Iowa State
Tried and failed
Single-crystal X-ray diffraction structure determination applied to heavy metal carboxylate coordination complexes. Reason: Crystal twinning and high background scattering prevented resolving light hydrogen atom positions
Natural deep eutectic solvents (NADESs) for lead acid battery recycling · Imperial
Tried and failed
in situ high-temperature x-ray diffraction applied to short-range order-disorder phase transitions in alloys. Outcome: no signal. Reason: XRD lacked sensitivity to detect short-range ordering (k-state) causing calorimetric specific heat anomalies
Thermophysical and Molten Salt Corrosion Behavior of Structural Materials for Next-Generation Clean Energy Systems · Georgia Tech
Tried and failed
X-ray diffraction phase analysis applied to deformation-induced retained austenite transformation. Outcome: no signal. Reason: Diffractometry was inconclusive in detecting retained austenite to martensite transformation in 300M steel.
Mechanical fragility, environmental stress, and radiation damage degrade crystal quality during handling
Crystals frequently cracked or deformed under mechanical handling, while room-temperature conditions caused rapid dehydration and radiation damage. In other experiments, cryoprotectant soaking and ligand binding disrupted crystal packing lattices, resulting in loss of diffraction or streaked patterns.
Tried and failed
post-crystallization soaking with standard cryoprotectants applied to protein crystals for X-ray diffraction. Reason: cryoprotectant soaking degraded crystal quality, causing low-resolution streaking diffraction
Structural Insights into Mycobacteriales Galactan Biosynthesis · MIT
Tried and failed
Ligand soaking and co-crystallization applied to protein crystal structure determination. Outcome: unstable. Reason: Ligand binding disrupted crystal packing, causing loss of X-ray diffraction
Characterization and targeting of the 2-methylcitrate cycle in Pseudomonas aeruginosa · Cambridge
Tried and failed
Protein X-ray crystallography applied to mutant protein structure determination. Reason: cysteine residues formed an artifactual intermolecular disulfide bond during crystal packing
Covalent Inhibition of Pro-Apoptotic BAX · Harvard
Considered and rejected
Considered and rejected: Shelved X-ray crystallography of the RecA3x-LexA complex after initial hits failed to yield interpretable diffraction due to ice crystal formation during harvesting, pivoting entirely to cryo-EM
BIOCHEMICAL AND STRUCTURAL ANALYSIS OF THE SOS SIGNAL COMPLEX · Penn
Considered and rejected
Considered and rejected: Discarded full-length wild-type PYROXD1 crystallization efforts after persistent crystal desiccation, radiation damage, and lack of usable diffraction
A study of select disparate causes of limb girdle muscular dystrophy - type 2/R · Texas Tech
Tried and failed
single-crystal X-ray diffraction applied to fragile organic molecular crystals. Outcome: unstable. Reason: Crystals were exceptionally brittle and bent or broke upon the slightest manipulation
Tried and failed
room-temperature single-crystal X-ray diffraction applied to light-sensitive protein crystals. Outcome: unstable. Reason: rapid dehydration, radiation damage, and poor diffraction quality under ambient conditions
Understanding Light-Regulation by CRYPTOCHROME: A Structural Story · Cornell
Tried and failed
inelastic x-ray scattering phonon dispersion measurement applied to organic and hybrid crystal acoustic phonons. Outcome: unstable. Reason: X-ray beam damage caused a 0.5% lattice parameter shift during measurement
EXPLORING THERMAL TRANSPORT IN ORGANIC AND HYBRID MATERIALS · Cornell
Peak overlap, severe peak asymmetry, and multiphase complexity hinder Rietveld refinement and phase identification
Diffraction patterns from multiphase mixtures and structurally similar intermetallic phases exhibited severe peak overlap that prevented convergence of Rietveld refinement. Thermal lattice distortions and decompression induced peak broadening and asymmetry, obscuring phase assignment and limiting quantitative analysis.
Tried and failed
single-crystal X-ray diffraction structure refinement applied to organic cocrystal hydrates. Outcome: did not converge. Reason: poor crystal diffraction quality prevented low R-factor refinement of all atoms
Lost to a baseline
Empirical Spurr and Myers XRD formula deviated by up to 20% compared to full-profile Rietveld refinement on nanocrystalline multi-phase powder samples.
Understanding the anatase-rutile stability in flame-made TiO 2 · Cambridge
Tried and failed
powder X-ray diffraction phase identification applied to biomass-derived complex multiphase chars. Reason: complex sharp diffraction peaks prevented assignment of specific crystalline inorganic phases
Understanding the relationship between synthetic conditions, porosity and CO2 uptake capacity of turbostratic carbons · University of Nottingham Repository
Tried and failed
Rietveld refinement of powder diffraction data applied to gas-loaded porous framework at low temperature. Outcome: did not converge. Reason: substantial peak asymmetry in low-temperature powder X-ray diffraction patterns
Tried and failed
powder x-ray diffraction phase identification after decompression applied to quenched high-pressure metastable intermetallic mixtures. Outcome: no signal. Reason: decompression caused broad diffraction peaks that prevented uniquely distinguishing and assigning recovered phases
Tried and failed
Rietveld refinement of X-ray diffraction patterns applied to quantifying structurally similar intermetallic phases. Outcome: no signal. Reason: diffraction peaks of the two candidate phases overlapped significantly
Process-microstructure-property relationships of high-pressure die cast AE44 magnesium alloy · Imperial
Tried and failed
Rietveld refinement of diffraction patterns applied to multiphase crystal structures. Outcome: did not converge. Reason: large number of phases and high degree of peak overlap prevented successful refinement
Design of Superelastic Secondary-Phase-Toughened Alloys · MIT
Synthetic coordination complexes and organic compounds fail to form suitable single crystals
Chemical synthesis of metal complexes, salts, and conjugated monomers repeatedly produced polycrystalline precipitates or solids of low crystallinity. Extensive crystallization attempts failed to yield single crystals large and ordered enough for single-crystal X-ray diffraction experiments.
Tried and failed
single-crystal X-ray diffraction applied to heterometallic coordination complexes. Outcome: no signal. Reason: Low crystallinity of solid-state complexes prevented determination of crystal structures
Multi-Stimuli-Responsive Network of Multicatalytic Reactions using a Single Palladium/Platinum Catalyst. · Cambridge
Tried and failed
single crystal growth for x-ray diffraction applied to coordination complex. Reason: repeated attempts failed to yield diffraction-quality crystals
Tried and failed
Single-crystal growth for X-ray diffraction applied to functionalized conjugated organic monomers. Reason: Crystals produced were of insufficient quality for single-crystal X-ray diffraction analysis
Considered and rejected
Considered and rejected: Rejected direct single-crystal X-ray diffraction for structure solution because large, high-quality single crystals could not be grown for metastable borides.
Unconventional synthesis of novel borides: interplay between crystal structures, magnetic and catalytic properties · Iowa State
Tried and failed
single-crystal growth for X-ray diffraction applied to doubly oxidized iron-sulfur cluster complex. Reason: crystals of sufficient quality for X-ray diffraction could not be obtained
Investigations of Iron–Nitrogen Bonding at Synthetic Iron–Sulfur Clusters · MIT
Tried and failed
single-crystal growth for X-ray diffraction applied to polyquaternary ammonium salt derivatives. Reason: Failed to form diffraction-quality single crystals despite multiple conditions
Total Synthesis of Oligomeric Cyclotryptamine Alkaloids · MIT
Tried and failed
Single crystal growth for X-ray diffraction applied to copper coordination complex. Reason: Crystallization attempts yielded only polycrystalline material instead of single crystals
Chemical Sensing of N-Nitrosodimethylamine and Methane · MIT
Preferred orientation, sample morphology, and lattice disorder distort diffraction intensities
Anisotropic crystal shapes and preferential grain alignment in solid pellets produced severe deviations in diffraction peak intensities away from random powder standards. Furthermore, complex stacking arrangements, overlapping unindexable lattices, and concurrent positional and chemical disorder compromised crystallographic modeling.
Tried and failed
macromolecular X-ray crystallography applied to protein-RNA complex. Reason: crystals exhibited multiple unindexable diffraction lattices
Structural Understanding of Remodeling Function of HIV-1 Reverse Transcriptase During Initiation · Harvard
Considered and rejected
Considered and rejected: Bulk powder X-ray diffraction patterns as the null signal for preferred orientation analysis, rejected because particle shape alters intrinsic peak intensities.
Considered and rejected
Considered and rejected: Rejected single-crystal XRD refinement modeling of both positional and chemical disorder simultaneously in M-TCPB tetramers due to space group constraints and refinement instability
Computational Modeling of Energetic and Diffusion Properties in Metal-Organic Frameworks · Georgia Tech
Considered and rejected
Considered and rejected: Rejected Williamson-Hall analysis on XRD profiles because PCL did not possess a sufficient number of diffraction peaks to yield an accurate deconvolution of crystal size and microstrain.
Microstructural evolution of electrospun microfibers for biomedical applications · Oxford
Tried and failed
direct powder-to-sintered pellet XRD pattern matching applied to sintered polycrystalline ceramic pellets. Outcome: did not generalise. Reason: preferential grain orientation and inhomogeneous phase distribution altered diffraction peak intensities
The Na|NaSICON interface in sodium solid-state batteries · Imperial
Considered and rejected
Considered and rejected: Rietveld XRD refinement rejected because heterogeneous serpentine ore forms sheet/cylinder structures violating random crystal orientation assumptions
Predicting Atmospheric CO2 Sequestration in Ultramafic Mine Tailings using an Empirically-Derived Model · Queens University Institutional Repository
Left open by the authors
Problems the authors named and did not get to.
Left open
Crystallize and solve the X-ray crystal structure of the dimeric extracellular domain of mincle. Blocker: Requires wet-lab protein purification, crystallization trials, and X-ray diffraction facilities
Left open
Structurally characterize intact BsuMI and LlaI R1-R2 or R1:R2:R3 protein complexes using X-ray crystallography or cryo-EM. Blocker: Requires a wet lab, protein expression/purification, and access to X-ray crystallography or cryo-EM facilities.
Left open
Solve the ternary complex structure of CRBN/FPFT-2216/PPIL4 using cryo-EM or X-ray crystallography. Blocker: Requires wet lab facilities, protein expression/purification, and cryo-EM or X-ray crystallography equipment.
Left open
Determine the 3D crystal structure of human NaCT experimentally using X-ray crystallography or cryo-EM. Blocker: Requires wet-lab facilities, protein expression/purification, and X-ray crystallography or cryo-EM equipment
Biological consequences of NaCT/SLC13A5/mINDY deffiency: Good versus evil, separated only by the blood-brain barrier · Texas Tech
Left open
Resolve WCC-C-box crystal structures using the identified minimal DNA binding sequences or cryo-EM. Blocker: Requires wet lab facilities, protein/DNA sample preparation, and X-ray crystallography or cryo-EM equipment.
THE NEUROSPORA CRASSA CIRCADIAN CLOCK: AN ORDERLY LOOP DRIVEN BY A DISORDERED CONDUCTOR · Cornell
Left open
Determine the membrane binding mechanisms and oligomerization states of BcLOV4 using cryo-electron microscopy or X-ray crystallography. Blocker: Requires a wet lab, protein purification, and structural biology experimental apparatus (cryo-EM/X-ray crystallography)
Biophysical Dynamics Of Rgs-Lov Proteins As Systems For Light-Induced Membrane Recruitment · Penn
Left open
Determine the structure of the Zpr1-eEF1A-Aim29-GTP-γ-S quaternary complex using cryo-EM or X-ray crystallography. Blocker: Requires a structural biology wet lab with access to cryo-EM or crystallography facilities and protein expression/purification
The tailored chaperone pathway for eukaryotic elongation factor 1A (eEF1A) biogenesis · Harvard
Left open
Determine high-resolution cryo-EM or X-ray crystal structures of cytotoxic TG2 forms, including the R580K open state dimer and inhibitor complexes. Blocker: Requires wet-lab protein expression, purification, crystallization/cryo-EM grid preparation, and access to X-ray beamlines or cryo-EM facilities
BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF G PROTEIN-COUPLED RECEPTOR ASSOCIATED ENZYMES · Cornell
Left open
Perform structural characterisation of UapA-WT or UapA-Q408EΔ1-11 bound to 3-benzylxanthine using cryo-EM and X-ray crystallography. Blocker: Requires wet lab facilities, purified protein samples, chemical analogues, and cryo-EM/crystallography equipment
Left open
Resolve the crystal structure of MAB_2736c bound to the mycobacterial ribosome using X-ray crystallography or cryo-EM. Blocker: Requires wet lab facilities for protein/ribosome purification and X-ray crystallography or cryo-EM structural determination
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