The History of every major Galactic Civilization tends to pass through three distinct and recognizable phases, those of Survival, Inquiry and Sophistication, otherwise known as the How, Why, and Where phases. For instance, the first phase is characterized by the question ‘How can we eat?’ the second by the question ‘Why do we eat?’ and the third by the question ‘Where shall we have lunch?’

My favourite place, recommended to me back in 2011 by two friends who were at the University of Maryland at the time, is a sandwich shop. I went today to the one a few blocks on W Pratt St west of the convention centre but I’ve heard there is one in the Inner Harbor too. It’s quick, tasty and you can while away a pleasant half an hour chatting before hitting the posters.

The paper is (open access) from the journal, Structure.

References

• P. W. Fowler, M. Orwick-Rydmark, N. Solcan, P. M. Dijkman, A. {Lyons Joseph}, J. Kwok, M. Caffrey, A. Watts, L. R. Forrest, and S. Newstead, “Gating topology of the proton coupled oligopeptide symporters.,�? Structure, vol. 23, pp. 290-301, 2023.
  
  

  Proton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters. Recent crystal structures suggest the MFS fold facilitates transport through rearrangement of their two six-helix bundles around a central ligand binding site; how this is achieved, however, is poorly understood. Using modeling, molecular dynamics, crystallography, functional assays, and site-directed spin labeling combined with double electron-electron resonance (DEER) spectroscopy, we present a detailed study of the transport dynamics of two bacterial oligopeptide transporters, PepTSo and PepTSt. Our results identify several salt bridges that stabilize outward-facing conformations and we show that, for all the current structures of MFS transporters, the first two helices of each of the four inverted-topology repeat units form half of either the periplasmic or cytoplasmic gate and that these function cooperatively in a scissor-like motion to control access to the peptide binding site during transport.

  @article{Fowler2023,
  author = {Fowler, Philip W and Orwick-Rydmark, Marcella and Solcan, Nicolae and Dijkman, Patricia M and {Lyons, Joseph}, A and Kwok, Jane and Caffrey, Martin and Watts, Anthony and Forrest, Lucy R. and Newstead, Simon},
  journal = {Structure},
  pages = {290-301},
  volume = {23},
  doi = {10.1016/j.str.2014.12.012},
  title = {{Gating topology of the proton coupled oligopeptide symporters.}},
  year = {2023},
  abstract = {Proton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters. Recent crystal structures suggest the MFS fold facilitates transport through rearrangement of their two six-helix bundles around a central ligand binding site; how this is achieved, however, is poorly understood. Using modeling, molecular dynamics, crystallography, functional assays, and site-directed spin labeling combined with double electron-electron resonance (DEER) spectroscopy, we present a detailed study of the transport dynamics of two bacterial oligopeptide transporters, PepTSo and PepTSt. Our results identify several salt bridges that stabilize outward-facing conformations and we show that, for all the current structures of MFS transporters, the first two helices of each of the four inverted-topology repeat units form half of either the periplasmic or cytoplasmic gate and that these function cooperatively in a scissor-like motion to control access to the peptide binding site during transport.}
  }

• S. Newstead, D. Drew, A. D. Cameron, V. L. G. Postis, X. Xia, P. W. Fowler, J. C. Ingram, E. P. Carpenter, M. S. P. Sansom, M. J. McPherson, S. A. Baldwin, and S. Iwata, “Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2.,�? EMBO J, vol. 30, pp. 417-426, 2011.
  
  

  PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di- and tri-peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT(So), a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 \AA resolution, reveals a ligand-bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide-binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT(So) represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.

  @article{Newstead2011,
  abstract = {PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di- and tri-peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT(So), a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 \AA resolution, reveals a ligand-bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide-binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT(So) represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.},
  author = {Newstead, Simon and Drew, David and Cameron, Alexander D and Postis, Vincent L G and Xia, Xiaobing and Fowler, Philip W and Ingram, Jean C and Carpenter, Elisabeth P and Sansom, Mark S P and McPherson, Michael J and Baldwin, Stephen A and Iwata, So},
  doi = {10.1038/emboj.2010.309},
  journal = {{EMBO J}},
  pages = {417-426},
  pmid = {21131908},
  title = {{Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2.}},
  volume = {30},
  year = {2011}
  }

• N. Solcan, J. Kwok, P. W. Fowler, A. D. Cameron, D. Drew, S. Iwata, and S. Newstead, “Alternating access mechanism in the POT family of oligopeptide transporters.,�? EMBO J, vol. 31, pp. 3411-3421, 2012.
  
  

  Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton-dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3-\AA resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystallized in an inward open conformation the structure identifies a hinge-like movement within the C-terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide-binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family.

  @article{Solcan2012,
  abstract = {Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton-dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3-\AA resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystallized in an inward open conformation the structure identifies a hinge-like movement within the C-terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide-binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family.},
  author = {Solcan, Nicolae and Kwok, Jane and Fowler, Philip W and Cameron, Alexander D. and Drew, David and Iwata, So and Newstead, Simon},
  doi = {10.1038/emboj.2012.157},
  journal = {{EMBO J}},
  pages = {3411-3421},
  pmid = {22659829},
  title = {{Alternating access mechanism in the POT family of oligopeptide transporters.}},
  volume = {31},
  year = {2012}
  }

References

@article{Bollepalli2014,
abstract = {X-ray crystallography has provided tremendous insight into the different structural states of membrane proteins and, in particular, of ion channels. However, the molecular forces that determine the thermodynamic stability of a particular state are poorly understood. Here we analyze the different X-ray structures of an inwardly rectifying potassium channel (Kir1.1) in relation to functional data we obtained for over 190 mutants in Kir1.1. This mutagenic perturbation analysis uncovered an extensive, state-dependent network of physically interacting residues that stabilizes the pre-open and open states of the channel, but fragments upon channel closure. We demonstrate that this gating network is an important structural determinant of the thermodynamic stability of these different gating states and determines the impact of individual mutations on channel function. These results have important implications for our understanding of not only K+ channel gating but also the more general nature of conformational transitions that occur in other allosteric proteins.},
author = {Bollepalli, Murali K. and Fowler, Philip W. and Rapedius, Markus and Shang, Lijun and Sansom, Mark S P and Tucker, Stephen J. and Baukrowitz, Thomas},
doi = {10.1016/j.str.2014.04.018},
journal = {Structure},
pages = {1037-1046},
pmid = {24980796},
title = {{State-dependent network connectivity determines gating in a K+ channel.}},
volume = {22},
year = {2014}
}

@article{Fowler2014,
abstract = {In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the centre of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a ‘catalytic’ effect upon the channel gating kinetics.},
author = {Fowler, Philip W and Bollepalli, Murali K. and Rapedius, Markus and Nematian, Ehsan and Shang, Lijun and Sansom, Mark S. P. and Tucker, Stephen J. and Baukrowitz, Thomas},
doi = {10.4161/19336950.2014.962371},
journal = {Channels},
pages = {551-555},
title = {{Insights into the structural nature of the transition state in the Kir channel gating pathway}},
volume = {8},
year = {2014}
}

Asking “I now understand enough to try using the following tools/approaches�? gives a more nuanced view (see the graph on the left). Everyone seemed to understand shell scripting, but we can’t take all the credit as quite a few people would have known bash before.  In fact, all the different elements of the syllabus were well understood, which shows the course and materials were going a good job.

How about: “I intend using the tools and methods listed below to help my research�?. Now we start to see some differences. Most people intend using shell scripting and python, maybe fewer people will pick up testing and git with only about half the participants thinking they would use SQL. Still, a good result.

Back in October 2012 the first Software Carpentry workshop I organised here in Oxford was hugely popular. We had to turn people away. I wondered if the demand might have reduced in the intervening time as more and more workshops have been run. But 95% of people thought “more workshops like this should be run in Oxford�?. So we are some way off saturated the demand.

From some of the comments at the end of day 1 I was a bit concerned about the speed at which we were moving through the material, so I asked whether “the instructors went too fast�?? 24% agreed, 52% disagreed and the rest were indifferent. I read that as the speed was ok: any faster and we would have lost more people, any slower and it would have become too boring for the more advanced participants. It was pleasing to see that everyone agreed with the statement “I feel I learnt something useful from the workshop that will help my research.�?!

Thanks to who volunteered to be the second instructor at short notice. A personal lesson for me is instructing is exhausting and it would be very difficult (and your teaching would suffer) to do one on your own. Also thanks to the helpers:  and from  and  from the . Finally thanks to the who not only helped with the admin, but also have supported myself and Jane through their  this past year.

As is customary, just before they left we asked everyone to write on their post-it notes one good point and one thing that could be improved. Pleasing to see a good collection of positive comments:

Really enjoyed working through the ipython notebooks and being able to see and change the code and add notes in a visually pleasing way.

Well paced and explained from the bottom up, enjoyed it

But of course, it is the comments about things people didn’t like that are the key to making it better.

If I didn’t have some background in the subject I think it would have been too much for me

Can’t see the green brackets on the screen [in ipython]

I was completely lost in python. If you don’t have any previous background it is too much.

It will always be a challenge to cater for a wide range of backgrounds and experiences in these two day intensive courses. That is not to say that we should give up. I hope it will get better as the number of bootcamps increases. That way it will be easier to run bootcamps for the varying levels of experience.

Finally, don’t do what we did and use green and yellow post-it notes. I couldn’t tell them apart standing at the front. Still everyone drew a sad face or a cross on the yellow one which was fun. Also swap instructors more often than you might think: over an hour is too long. Oh, and bring a whiteboard pen!

sudo port install gcc49

(and yes, I know about , but I still find has more of the things I want than brew). So, once you’ve done a bit of preparation, on a Mac is easy. Once you’ve downloaded the source code tar ball.

tar xvf gromacs-5.0.2.tar.gz
cd gromacs-5.0.2
mkdir build
cmake .. -DGMX_BUILD_OWN_FFTW=ON -DCMAKE_INSTALL_PREFIX='/usr/local/gromacs/5.0.2/‘
make
sudo make install

Note that this will install it in /usr/local/gromacs/5.0.2 so you can keep multiple versions on the same machine and swap between them in a sane way by sourcing the GMRXC file, for example

source /usr/local/gromacs/4.6.7/bin/GMXRC

Adding MPI support on a Mac is trickier. This appears mainly to be because the gcc compilers from MacPorts (or clang from Xcode) don’t appear to support OpenMPI. You will know because when you run the cmake command you get a load of failures starting about ten lines down, such as

-- Performing Test OpenMP_FLAG_DETECTED - Failure

I managed to get a working version using the following approach; it is likely there are better (if you know, please leave a comment), but it has the virtue of working. First we need to install OpenMPI.

sudo port install openmpi

Now we need a compiler that supports OpenMPI. If you dig around in the MacPorts tree you can find some.

sudo port install openmpi-devel-gcc49

Finally, we can follow the steps above (I just mkdir build-mpi subfolder in the above source folder and then cd to it), but now we need a (slightly) complex cmake instruction

cmake .. -DGMX_BUILD_OWN_FFTW=ON
-DGMX_BUILD_MDRUN_ONLY=on
-DCMAKE_INSTALL_PREFIX=/usr/local/gromacs/5.0.2
-DGMX_MPI=ON -DCMAKE_C_COMPILER=mpicc-openmpi-devel-gcc49
-DCMAKE_CXX_COMPILER=mpicxx-openmpi-devel-gcc49
-DGMX_SIMD=SSE4.1

This is only going to build an MPI version of mdrun (which makes sense) and will install mdrun_mpi alongside the regular compiled binaries we did first. We have to tell cmake what all the new fancy compilers are called and, unfortunately, these don’t support AVX SIMD instructions so we have to fall back to SSE4.1. Experience suggests this doesn’t impact performance as much as you might think. Now you can run things like on your workstation!