Scott Classen

Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)

We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.

Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair

The Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT(1)-BRCT(2) domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a bipartite MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs.

XRCC4 Protein Interactions with XRCC4-like Factor (XLF) Create an Extended Grooved Scaffold for DNA Ligation and Double Strand Break Repair.

The XRCC4-like factor (XLF)-XRCC4 complex is essential for nonhomologous end joining, the major repair pathway for DNA double strand breaks in human cells. Yet, how XLF binds XRCC4 and impacts nonhomologous end joining functions has been enigmatic. Here, we report the XLF-XRCC4 complex crystal structure in combination with biophysical and mutational analyses to define the XLF-XRCC4 interactions. Crystal and solution structures plus mutations characterize alternating XRCC4 and XLF head domain interfaces forming parallel super-helical filaments. XLF Leu-115 (“Leu-lock”) inserts into a hydrophobic pocket formed by XRCC4 Met-59, Met-61, Lys-65, Lys-99, Phe-106, and Leu-108 in synergy with pseudo-symmetric β-zipper hydrogen bonds to drive specificity. XLF C terminus and DNA enhance parallel filament formation. Super-helical XLF-XRCC4 filaments form a positively charged channel to bind DNA and align ends for efficient ligation. Collective results reveal how human XLF and XRCC4 interact to bind DNA, suggest consequences of patient mutations, and support a unified molecular mechanism for XLF-XRCC4 stimulation of DNA ligation.

Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source

The SIBYLS beamline of the Advanced Light Source at Lawrence Berkeley National Laboratory is a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline. Key features and capabilities are described along with implementation and performance.

High-throughput SAXS for the characterization of biomolecules in solution: a practical approach.

The recent innovation of collecting X-ray scattering from solutions containing purified macromolecules in high-throughput has yet to be truly exploited by the biological community. Yet, this capability is becoming critical given that the growth of sequence and genomics data is significantly outpacing structural biology results. Given the huge mismatch in information growth rates between sequence and structural methods, their combined high-throughput and high success rate make high-throughput small angle X-ray scattering (HT-SAXS) analyses increasingly valuable. HT-SAXS connects sequence as well as NMR and crystallographic results to biological outcomes by defining the flexible and dynamic complexes controlling cell biology. Commonly falling under the umbrella of bio-SAXS, HT-SAXS data collection pipelines have or are being developed at most synchrotrons. How investigators practically get their biomolecules of interest into these pipelines, balance sample requirements and manage HT-SAXS data output format varies from facility to facility. While these features are unlikely to be standardized across synchrotron beamlines, a detailed description of HT-SAXS issues for one pipeline provides investigators with a practical guide to the general procedures they will encounter. One of the longest running and generally accessible HT-SAXS endstations is the SIBYLS beamline at the Advanced Light Source in Berkeley CA. Here we describe the current state of the SIBYLS HT-SAXS pipeline, what is necessary for investigators to integrate into it, the output format and a summary of results from 2 years of operation. Assessment of accumulated data informs issues of concentration, background, buffers, sample handling, sample shipping, homogeneity requirements, error sources, aggregation, radiation sensitivity, interpretation, and flags for concern. By quantitatively examining success and failures as a function of sample and data characteristics, we define practical concerns, considerations, and concepts for optimally applying HT-SAXS techniques to biological samples.

Software for the high-throughput collection of SAXS data using an enhanced Blu-Ice/DCS control system

The Blu-Ice GUI and Distributed Control System (DCS) developed in the Macromolecular Crystallography Group at the Stanford Synchrotron Radiation Laboratory has been optimized, extended and enhanced to suit the specific needs of the SAXS endstation at the SIBYLS beamline at the Advanced Light Source. The customizations reported here provide one potential route for other SAXS beamlines in need of robust and efficient beamline control software.

An atomic-resolution view of neofunctionalization in the evolution of apicomplexan lactate dehydrogenases.

Malate and lactate dehydrogenases (MDH and LDH) are homologous, core metabolic enzymes that share a fold and catalytic mechanism yet possess strict specificity for their substrates. In the Apicomplexa, convergent evolution of an unusual LDH from MDH produced a difference in specificity exceeding 12 orders of magnitude. The mechanisms responsible for this extraordinary functional shift are currently unknown. Using ancestral protein resurrection, we find that specificity evolved in apicomplexan LDHs by classic neofunctionalization characterized by long-range epistasis, a promiscuous intermediate, and few gain-of-function mutations of large effect. In canonical MDHs and LDHs, a single residue in the active-site loop governs substrate specificity: Arg102 in MDHs and Gln102 in LDHs. During the evolution of the apicomplexan LDH, however, specificity switched via an insertion that shifted the position and identity of this ‘specificity residue’ to Trp107f. Residues far from the active site also determine specificity, as shown by the crystal structures of three ancestral proteins bracketing the key duplication event. This work provides an unprecedented atomic-resolution view of evolutionary trajectories creating a nascent enzymatic function. DOI: http://dx.doi.org/10.7554/eLife.02304.001

The R‐factor gap in macromolecular crystallography: an untapped potential for insights on accurate structures

In macromolecular crystallography, the agreement between observed and predicted structure factors (Rcryst and Rfree) is seldom better than 20%. This is much larger than the estimate of experimental error (Rmerge). The difference between Rcryst and Rmerge is the R‐factor gap. There is no such gap in small‐molecule crystallography, for which calculated structure factors are generally considered more accurate than the experimental measurements. Perhaps the true noise level of macromolecular data is higher than expected? Or is the gap caused by inaccurate phases that trap refined models in local minima? By generating simulated diffraction patterns using the program MLFSOM, and including every conceivable source of experimental error, we show that neither is the case. Processing our simulated data yielded values that were indistinguishable from those of real data for all crystallographic statistics except the final Rcryst and Rfree. These values decreased to 3.8% and 5.5% for simulated data, suggesting that the reason for high R‐factors in macromolecular crystallography is neither experimental error nor phase bias, but rather an underlying inadequacy in the models used to explain our observations. The present inability to accurately represent the entire macromolecule with both its flexibility and its protein‐solvent interface may be improved by synergies between small‐angle X‐ray scattering, computational chemistry and crystallography. The exciting implication of our finding is that macromolecular data contain substantial hidden and untapped potential to resolve ambiguities in the true nature of the nanoscale, a task that the second century of crystallography promises to fulfill.

Functional and structural resilience of the active site loop in the evolution of Plasmodium lactate dehydrogenase

The malarial pathogen Plasmodium falciparum ( Pf) is a member of the Apicomplexa, which independently evolved a highly specific lactate dehydrogenase (LDH) from an ancestral malate dehydrogenase (MDH) via a five-residue insertion in a key active site loop. PfLDH is widely considered an attractive drug target because of its unique active site. The conservation of the apicomplexan loop suggests that a precise insertion sequence was required for the evolution of LDH specificity. Aside from a single critical tryptophan, W107f, the functional and structural roles of residues in the loop are currently unknown. Here we show that the loop is remarkably robust to mutation, as activity is resilient to radical perturbations of both loop identity and length. Thus, alternative insertions could have evolved LDH specificity as long as they contained a tryptophan in the proper location. PfLDH likely has great potential to develop resistance to drugs designed to target its distinctive active site loop.

Erratum: ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling (EMBO Journal (2014) 33 (482-500)) DOI 10.1002/embj.201386100

Thank you for submitting your manuscript on ATP-driven Rad50 conformational changes for consideration by The EMBO Journal. We have now received comments from three expert referees who evaluated the study. I am pleased to inform that all of them consider this work of interest, and for the most part well-conducted and well-presented, and we should therefore be happy to consider a revised version of this manuscript further for publication in our journal. Nevertheless, all referees do raise a number of specific issues that would need to be satisfactorily addressed prior to acceptance. As you will see from the reports, while this amounts to a considerable number of points, most of them appear to be well taken and straightforward, and referring to specific issues rather than major conceptual concerns. I would therefore like to invite you to revise the study taking into account the various referee comments and suggestions, and to resubmit a revised manuscript together with a detailed point-by-point response letter. In doing so, please also make sure to expand the discussion (as requested by referee 3) to better discuss your results in light of relevant previously published findings, including those in your own recent paper (Lee et al, JBC March 2013).