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Q: How do I unmount a disk device and remove its device association Come at this from the point of view of ransomware, where attached backup media could be corrupted, regardless of it being previously mounted.
I have some USB flash drives (hereafter "USBFD" or "USBFDs") which I perform offsite backups to daily. Several hours elapse between the time the backup script finishes writing to the USBFDs and when I pull the day's USBFD from the server. Theoretically, ransomware could look for devices in mount and /dev/. When I umount the USBFD via the script, can I also remove the /dev/sdX device as well so it can't be mounted again and the contents tampered with?
Something in the OS (ubuntu server) removes the /dev/sdX device after the drive is umount ed and pulled.
The USBFDs aren't in /etc/fstab; the UUIDs are in the script and they're mounted to the mountpoints using the partition UUID.
EDIT: To include example. At the end of the example below, the USBFD is still present in /dev/disk/by-* even though it's not mounted. I'd like to delete the device association /dev/sdb and /dev/sdb1 as though the drive has been pulled from the server.
$USER@U16:~$ ls -l /dev/disk/by-* #list all devices
/dev/disk/by-id:
...
lrwxrwxrwx 1 root root  9 Jan 17 06:39 usb-General_USB_Flash_Disk_0425640000007474-0:0 -> ../../sdb
...

/dev/disk/by-label:
...
lrwxrwxrwx 1 root root 10 Jan 17 06:39 ESD-USB -> ../../sdb1
...

/dev/disk/by-path:
...
lrwxrwxrwx 1 root root  9 Jan 17 06:39 pci-0000:00:10.1-usb-0:2:1.0-scsi-0:0:0:0 -> ../../sdb
lrwxrwxrwx 1 root root 10 Jan 17 06:39 pci-0000:00:10.1-usb-0:2:1.0-scsi-0:0:0:0-part1 -> ../../sdb1
...

/dev/disk/by-uuid:
...
lrwxrwxrwx 1 root root 10 Jan 17 06:39 9A95-F490 -> ../../sdb1
...

$USER@U16:~$ sudo umount /media/$USER/ESD-USB #umount the device @ mounpoint

$USER@U16:~$ ls -l /dev/disk/by-* #USBFD still shows in output although it's not mounted.
/dev/disk/by-id:
...
lrwxrwxrwx 1 root root  9 Jan 17 06:39 usb-General_USB_Flash_Disk_0425640000007474-0:0 -> ../../sdb
lrwxrwxrwx 1 root root 10 Jan 17 06:39 usb-General_USB_Flash_Disk_0425640000007474-0:0-part1 -> ../../sdb1
...

/dev/disk/by-label:
...
lrwxrwxrwx 1 root root 10 Jan 17 06:39 ESD-USB -> ../../sdb1
...

/dev/disk/by-path:
...
lrwxrwxrwx 1 root root  9 Jan 17 06:39 pci-0000:00:10.1-usb-0:2:1.0-scsi-0:0:0:0 -> ../../sdb
lrwxrwxrwx 1 root root 10 Jan 17 06:39 pci-0000:00:10.1-usb-0:2:1.0-scsi-0:0:0:0-part1 -> ../../sdb1
...

/dev/disk/by-uuid:
...
lrwxrwxrwx 1 root root 10 Jan 17 06:39 9A95-F490 -> ../../sdb1
...


We have sellers in Lees Summit who call us with a lot of different questions when they inherit or are going to be inheriting a property. In our last post, we discussed what probate is and when a property might need to go through probate.
Today we would like to look at what the probate process looks like and when can a house in probate be sold in Lees Summit.
First, an Executor needs to be named. This will usually be defined in a will. If there is no will the court will appoint an administrator for the estate. In some cases, we see this is also the attorney handling the probate for the estate.
Notify heirs and creditors of the probate proceedings.
Take possession of the assets of the deceased and prepare an inventory.
Collect all incomes due to the estate, such as interest, dividends, rents and other debts due.
If the deceased had a business, manage that and keep it going.
Take an inventory of all the heirs, who they are, where they are and how were they related.
If the deceased was party to a lawsuit, see that to completion.
Pay taxes and file final tax returns.
Pay all creditors of the deceased.
Sell assets to raise money to pay taxes and creditors and the attorney and CPA and other expenses of the estate.
Distribute assets to the heirs.
One of the duties is to sell assets to raise money or to divide the assets among the heirs and the question becomes when can those assets be sold? Contrary to popular opinion the house or houses in a probate case or other items of value can be sold during the probate period, but there is a process that must be followed. If you would rather not wait until probate is complete, you should consult with the probate attorney for the process of selling the property or assets during the process.
Roughly what needs to happen to sell during the process is as follows.
First: Determine the assets you want to sell.
Most often we deal with the house as the asset the heirs would like to sell. Assets that need to be sold could also be stocks, bonds, cars, collector's items, businesses or rental property.
Second: Determine the value of the asset.
Stocks and bonds are fairly easy to determine, other assets like houses require expert opinions of an appraiser. Different states have different laws, so if the house is believed to be under a certain value, it may be that the expert opinion of a Realtor would suffice without the expense of an Appraisal. We have seen this several times here in the KC metro. We have been asked our expert opinion on a house that was believed to be under $40,000 and as a small estate, worth less than $40,000 does not have to go through probate, it may be that a house worth less than $40,000 can sell without probate.
Third: Find a buyer for the asset.
When this asset is a house, you might want to sell directly to a cash buyer to speed up the process or hire a Realtor to maximize the sale price. You will need to weigh your options, which we will review in a future blog post. For other odd items like collectibles or businesses, you may also need to seek out an expert to help sell.
Fourth: Petition the court for approval of the sale.
In the case of assets under certain values, estates worth less than $40,000 for example, you may be able to sell without court approval. Other times with assets of larger values, like the house, you may need to get court approval. Please consult with your Probate Attorney.
Once you have a buyer with an agreed upon price and the approval of the court, you can then complete the sale. This usually means in Kansas and Missouri working with a local title company to manage the sale and the executor and the buyer signing off on all the closing documents. The buyer gets the asset or house and the estate gets the proceeds to split or to hold in the bank to wait out the rest of the probate process.
It is important to realize that some assets have holding costs and to consider the asset itself.
In the case of a house, it might be financially sound for the executor or an heir to live in the house and pay the holding costs of taxes, insurance, mortgage, utilities throughout all or some of the process, especially if the house is going to be distributed to the heir. But if no one is going to live in the house, and it is ultimately going to be sold, you need to look beyond all those holding costs. There will be other costs because the home is vacant. Insurance on a vacant property is much more expensive than for an owner-occupied home. And because it is a vacant property, someone needs to check on the home from time to time to make sure no one has moved in, there has been no vandalism, the pipes and other items have not frozen, that the snow is removed, and the yard is mowed. It is much less expensive to sell the house and probate the cash sitting in a bank over paying all the holding costs and overseeing the house if the house is going to sit vacantly.
Professional Home Buyers like Scott Tucker here at kcmoHomeBuyer specialize in helping people with homes that need to be sold during or after the probate process. Professional Home Buyers will look at the house and make an offer. This offer can be based on a house as it sits, needing all repairs and updates and can include leaving behind all the belongings or can include the heirs cleaning out the house. This offer can close quickly as the seller has no repairs to make and because the buyer does not have to wait for loan approval or it can close on the time frame of the heirs and the probate court. The average home buyer will not have the luxury of time and money like the professional home buyer and the average home buyer may not be willing to deal with property that is in need of major renovation or property that has tenants, should the estate hold rental property.
If you would like to sell an inherited property and would like to see what the Tuckers might offer, please fill out our form about your house or give us a call at 816-800-9044!
Words cannot express how Kim's hard work has affected my family. After a death in my family, I was left with a home out of state that had been abandoned and no banks would even claim the loan! Kim helped me figure out how to determine ownership, what to do about the taxes, and how to clear up the loan status so the property could be sold. She helped me through the process every step of the way, despite my frazzled mindset after losing a loved one. There is no one better, she's simply the best!


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James, Love, Irving make a formidable Cleveland Big 3
By David Simon | Sunday, August 24, 2014, 12:45 a.m.
Lay days, lay days go away, come again another day when the waiting periods have expired and Chopes isn't in contest mode and a six-star women's event isn't in its final rounds.
OK, I know that doesn't rhyme. Shakespeare, I am not.
Both the Billabong Pro Tahiti and the Swatch Girls Pro France had lay days on Friday and Saturday with hopes for action to resume today. Sebastian Zietz will be taking on John John Florence in the third round at Teahupoo when the men's World Championship Tour event resumes, while Tatiana Weston-Webb has her Round of 12 matchup against Laura Enever and Nikki Van Dijk with the top two moving into the quarterfinals.
And since I haven't quite mastered the art of bending the space-time continuum (though I am working on it), I don't know if the Kauai Red Raiders picked up their first win of the season in Saturday night's tilt with Damien High School.
So today we're widening the spectrum a bit. The long-anticipated trade sending Kevin Love to the Cleveland Cavaliers became official on Saturday. The Minnesota Timberwolves sent their franchise power forward to LeBron James' new/old team in exchange for this year's No. 1 overall draft pick, Andrew Wiggins, as well as last year's No. 1 overall pick, Anthony Bennett. The T'Wolves also added forward Thaddeus Young from the Philadelphia 76ers, who got a first-round pick, along with Alexey Shved and Luc Richard Mbah a Moute from Minnesota.
Now with James, Love and Kyrie Irving in tow, the Cavs are the team to beat in the Eastern Conference. When James opted not to re-sign to the Miami Heat and instead returned home to Cleveland, he regained some goodwill and public relations points. This deal doesn't negate all that, but it does change the way I view Cleveland, and not in a positive way.
As discussion of this trade surfaced almost immediately after the NBA Draft, I hoped against my better judgement that Cleveland wouldn't make the deal. Part of the allure of watching James head back to the Cavs was to see how he and Wiggins could work together. Wiggins might be a great player. He might also be just an average NBA rotation player. We won't know for some time, but his ceiling seemed to be sky high with James as a running mate.
So not only does this deal send him to a Minnesota team where no young player has excelled since Kevin Garnett, it removes the best player in the game from being able to help his transition to the pros. Wiggins could become great in Minnesota, but who's going to be paying attention?
Had he been able to stay with Cleveland and try to help a young team with championship aspirations, all NBA fans would be able to "witness" his progression, just as we had with James more than 10 years ago.
But that rationale is a completely selfish one. I can't really make a sound argument for why the Cavs should not have made this deal. Trading potential for a proven commodity is almost always correct. Trading potential for a proven superstar is never incorrect. Love has established himself to many as a top-10 player in the league. Adding that type of player is extremely rare, especially in Cleveland, where premiere players don't flock unless they happened to grow up in Akron.
There aren't many situations where teams regret adding an all-star – perhaps MVP – caliber player. On the flip side, there aren't many situations where a team knows it has to trade its superstar and comes away with as great a package as Minnesota received. Usually a team gets about 60 cents on the dollar for a top talent. This time, the T'Wolves may have gotten a transcendent player, along with Bennett and future flexibility. These deals are rarely that lucrative, so kudos to them.
Cleveland will be the favorites in the East, especially with the horrific injury to Indiana's Paul George, but the Cavs are anything but a sure thing. The only player in Cleveland who really knows what it takes to win it all is James. He certainly thinks the team has a better chance with Love than Wiggins, but it sure would have been fun to watch just how high he could carry the (once) young Cavs.
David Simon can be reached at dsimon@thegardenisland.com.
Red Raiders look to rebound against ILH's Damien
Monarchs hold off Red Raiders 28-21

redpajama_set_name: RedPajamaCommonCrawl


OneCoWork Plaça Catalunya is the most recent opened location of the collaborative workspace. Set in the city centre of Barcelona, offering ideal transport links for those who need a well-connected office to easily move around with high-quality service. The innovation and spectacular design of the location keeps their Members inspired each and every day. OneCoWork is home to a vibrant community of entrepreneurs, startups, freelancers and corporates and we offer price plans to suit every business imaginable.
Agathe B.2019/02/1410Nothing better than working in a community such as the one in OneCoWork, a relaxing atmosphere that allows fruitful work! What about design ? Unbelievable !


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E3 2007: How Sony Saved E3 2007
July 11, 2007 Arnold-Katayev — No Comments
Before I get into the whole spiel with hands-on impressions, allow me to boldly state that Sony saved this year's E3. Call me whatever you want for saying this, but after Microsoft's terrible conference and Nintendo's snoozefest, Sony was the only company who had a conference worth watching. Bit after bit, it was exciting news all around. You had the revelation of WipeOut HD, the drool inducting new video of Gran Turismo, the absurdly awesome Metal Gear Solid 4 trailer, Killzone 2, Unreal Tournament III being a timed-exclusive, the slimmer PSP, and everything in between. The crowd was genuinely interested in everything Sony was saying, and the content we were treated to was definitely the best of any conference by far.
I was a little surprised that Sony made no mention of an all new vibrating PS3 controller, but perhaps they've got something up their sleeves and we'll have to wait a bit more until we see it. The best part about it all was that after the conference ended, to the right of the room was a door that allowed us to walk into the next stage. And that stage Sony called the arcade, and it allowed us to sample dozens of different games, both 1st and 3rd party. The arcade was terrific, and it was Sony's homage to the spirit of E3 and its grandiosity. There was plenty of kiosks to play on, and the game lineup featured all of Sony's featured first party titles for E3, in addition to surprises like Unreal Tournament III, Time Crisis 4, Sega Rally Revo, and others.
There is no doubt about it, but Sony stole this show with a landslide victory and if it wasn't for their solid conference and arcade game lounge thereafter, this may very well have been a disaster for me. Unfortunately, the selection of games for the entire show is quite slim. I got my hands on 18 games, and from there on, I didn't see much that I had to cover. At most, there are another 10-15 games out there for me to play (a lot of which are only displayed on an Xbox 360 kiosk), and that's a drastic difference compared to almost 100 from years before. Now with that out of the way, enjoy the impressions.


This summer, the Republican Party of Texas took a bold step forward on the issues of border security and immigration reform by adopting "The Texas Solution." The national Republican Party has now followed suit by adopting a guest worker program into its national platform. This has not gone un-noticed by the national media.
Most recently, the New York Times took note of this movement within the party and the adoption of this new platform plank. In an article titled, "A Republican Platform Line Friendly to Immigrants", writer Julia Preston gives credit to the Republican Party for moving forward on this issue.
Preston credits Republican immigration activist, Brad Bailey, with making this happen. Baily, who is a restaurant owner in the Houston area, was one of the architects of The Texas Solution and has since worked, along with others, tirelessly to get this plank added to the national platform.
Texas Republicans Malcolm Morris, Tom Mechler and Brad Bailey present the 2012 National Republican Party Platform containing the foreign guest-worker plank.
Bailey told the NY Times, "We are a border state and we have the same problem as Arizona, but we addressed it totally differently than Arizona." Indeed, Texas dodged a bullet by not following in the path of anti-immigrant legislation put forward by states like Arizona, Alabama and Georgia. All of these states have suffered economic loss because of their actions and many have taken steps to reverse course.
"Jan Brewer and Joe Arpaio are hijacking the issue and damaging the Republican brand, " Baily continued. "We need to stop the hatred language and fix this problem."
During the years I have been working on the problems of illegal immigration and border security, little has been done to actually move a solution forward. The result is a de facto amnesty that has left millions of non-identified illegal immigrants free to roam the country with no accountability. We need to establish a process of identifying all immigrants in our country. We need to know who is here and why and that is not being done.
Texas took the lead last year through a proposed resolution in the Texas Legislature. HCR 88 received broad based, bi-partisan support from legislators even though it never made it from the Calendars Committee to the floor for a House vote. This was followed by the RPT's adoption of The Texas Solution.
It is interesting that one of the main themes of this year's RNC Convention is the "We did built it" line referring to President Obama's slap in the face to small business owners across the county. Yet, when business owners make the claim that they cannot find a reliable source of manual labor, they are chastised by fellow Republicans for only wanting cheap labor. The business leaders I talk to are simply looking for labor, at any price. They cannot find it. When you have restaurants like the Chick-fil-A chain, now beloved by conservatives, who are paying over $10 per hour for labor and still have difficulty finding US citizens willing to apply for the jobs or do the work, it becomes very clear we do have a labor shortage issue. And this is also true in construction and other hospitality industries.
Last night, former Secretary of State, Condoleezza Rice, picked up on the new theme of the RNC Platform stating that Republicans need to champion a "compassionate" immigration policy. "We must continue to welcome the world's most ambitious people to be a part of us. In that way we stay young and optimistic and determined. We need immigration laws that protect our borders; meet our economic needs, and yet show that we are a compassionate people." Her statement was received enthusiastically by the delegates of the convention.
Obama's record on immigration reform is just one of his many failures. But it was not a failure because he tried something that didn't work. For three years, he did nothing. During two of those years he could have done anything he wanted, but still did nothing. And now, as a last ditch pandering attempt, he has created an illegal DREAM Act, bypassing our legislative process altogether.
Republicans will continue to lead on this issue. With a new Republican president and Republican-controlled Congress, we will be able to take action to positively secure our border and create a new, positive immigration program that accomplishes the goals set forth by Secretary Rice above. It is only the Republican Party that can effectively create a realistic and workable solution we all can live with. Democrats have proven they cannot and will not.
What about undocumented granny and auntie?
The proposed guest worker program is a step in the right direction, however, Latinos and other immigrants want to know what the GOP has in store for undocumented immigrants who are no longer part of the work force such as granny and auntie. They do not have employers that can sponsor them.
Will Romney-Ryan encourage them to self-deport and leave their grown-up children and grandchildren behind?
As long as the Republicans do not have a solution for granny and auntie, it will be hard to convince more Latinos and other immigrants to vote for them.


Best Five new Launches on Motorola One Power Android Versions Pie For the first time in the whole world.
Motorola One Power has updated 5 new futures in Android version Pie throughout the world, and it has only been done in Motorola One Power and it has happened with Android Versions Pie, and we will see it today in full detail.
Finally, the first Motorola One Power World received the long-awaited update to Android Versions Pie.
The system's new features are numerous, but of course, there are some functions that stand out from others.
I have separated the five features that Motorola One Power received with this update and what you need to know.
The most interesting feature of Android Versions Pie is still present in the Motorola One Power, which is Digital Wellness. Basically, Digital Wellness is a panel that brings some tools that aim to monitor the time spent by the user in social networks and with the cell phone.
The goal is to monitor and, at the end of the week, deliver reports on user morals.
Google removed from Motorola One Power the final version of Android Versions Pie the option that set the notch to disguise it, however, the dark mode remained present as one of the most awaited features of system fans. Unlike Android Oreo, in its native version, the themes of the Pie vary between light, dark and automatic (in the Oreo there was only automatic, which varied according to the color of the wallpaper).
What differences are there between Android Go and One?
Themes do not have transparency in the application drawer, as it was in Oreo. You find this option in the "Screen" menu, among the system settings.
Gesture navigation is disabled by default so the user needs to go to System> Gestures to enable navigation that replaces conventional buttons. The Home button now has the function of launching Google Assistant, scrolling to show open applications, and by sliding it up you access multitasking. The back button automatically appears in specific applications.
There was a lack of better integration of Google Lens with the Motorola One Power native camera.
In this version, users will be able to rely on Google's intelligent system that uses Augmented Intelligence to scan codes, labels, objects, and landscapes and display useful information such as pricing, addresses, and others.
Android Versions Pie's big difference is the new floating menus and the new fast shortcut center, which have been redesigned and are more practical and feature rounded icons.
These menus, such as sound control, for example, follow the pattern of the theme chosen by the user, but it is not possible to change the predominant emerald green color of the icons and the context of the interface.
The user must enter the settings of the home screen – clicking and pressing the home screen – and change.
The layout of icons between different formats, such as the tear, circle, square, square with rounded corners and others. The color scheme, however, will remain the native of the system.
So what Android Versions Pie feature do you like the most? Already received the update on your smartphone?
If you liked this article, please do not forget to comment and what is written in this article is true.


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Silent Snow, Secret Snow
by Conrad Aiken [?]
Just why it should have happened, or why it should have happened just when it did, he could not, of course, possibly have said; nor perhaps could it even have occurred to him to ask. The thing was above all a secret, something to be preciously concealed from Mother and Father; and to that very fact it owed an enormous part of its deliciousness. It was like a peculiarly beautiful trinket to be carried unmentioned in one's trouser-pocket-a rare stamp, an old coin, a few tiny gold links found trodden out of shape on the path in the park, a pebble of carnelian, a sea shell distinguishable from all others by an unusual spot or stripe-and, as if it were any one of these, he carried around with him everywhere a warm and persistent and increasingly beautiful sense of possession. Nor was it only a sense of possession-it was also a sense of protection. It was as if, in some delightful way, his secret gave him a fortress, a wall behind which he could retreat into heavenly seclusion. This was almost the first thing he had noticed about it-apart from the oddness of the thing itself-and it was this that now again, for the fiftieth time, occurred to him, as he sat in the little schoolroom. It was the half hour for geography. Miss Robinson was revolving with one finger, slowly, a huge terrestrial globe which had been placed on her desk. The green and yellow continents passed and re-passed, questions were asked and answered, and now the little girl in front of him, Astrith, who had a funny little constellation of freckles on the back of her neck, exactly like the Big Dipper, was standing up and telling Miss Robinson that the equator was the line that ran round the middle.
Miss Robinson's face, which was old and grayish and kindly, with gray stiff curls beside the cheeks, and eyes that swam very brightly, like little minnows, behind thick glasses, wrinkled itself into a complication of amusements.
"Ah! I see. The earth is wearing a belt, or a sash. Or someone drew a line around it!"
"Oh, no-not that-I mean"-
In the general laughter, he did not share, or only a very little. He was thinking about the Arctic and Antarctic regions, which of course, on the globe, were white. Miss Robinson was now telling them about tropics, the jungles, the steamy heat or equatorial swamps, where the birds and butterflies, and even the snakes, were like living jewels. As he listened to these things, he was already, with a pleasant sense of half-effort, putting his secret between himself and the words. Was it really an effort at all? For effort implied something voluntary, and perhaps even something one did not especially want; whereas this was distinctly pleasant, and came almost of its own accord. All he needed to do was to think of that morning, the first one, and then of all the others-
But it was all so absurdly simple! It had amounted to all so little. It was nothing, just an idea-and just why it should have become so wonderful, so permanent, was a mystery-a very pleasant one, to be sure, but also, in an amusing way, foolish. However, without ceasing to listen to Miss Robinson, who had now moved up to the north temperate zone, he deliberated his memory of the first morning. It was only a moment or two after he had awakened-or perhaps the moment itself. But was there, to be exact, an exact moment? Was one awake all at once? Or was it gradual? Anyway, it was after he had stretched a lazy hand up towards the head rail, and yawned, and then relaxed again among his warm covers, all the more grateful on a December morning, that the thing had happened. Suddenly, for no reason, he had thought of the postman, he remembered the postman. Perhaps there was nothing so odd in that. After all, he heard the postman almost every morning in his life-his heavy boots could be heard clumping round the corner at the top of the little cobbled hill-street, and then, progressively nearer, progressively louder, the double knock at each door, the crossings and re-crossings of the street, till finally the clumsy steps came stumbling across to the very door, and the tremendous knock came which shook the house itself.
Joel Chandler HarrisMr. Rabbit Meets His Match Again
George AdeThe New Fable Of The Marathon In The Mud And The Laurel…
Rex BeachThe Wag-Lady
James ThurberThe Catbird Seat
Ryunosuke AkutagawaIn a Grove
Dorothy ParkerYou Were Perfectly Fine


Detention centers are spread throughout the United States. The Tacoma Dominicans have one in our "backyard," about 10 minutes from our administrative offices. On July 15, 15 sisters and associates spent the afternoon outside the detention center, offering refreshments and support to the families returning from visiting their loved ones. As they came through the gates, many in tears, we welcomed them, listened to them, and offered refreshment. It was a hot day, and many people had traveled hundreds of miles to visit a husband, brother, uncle or father. Children especially were distraught because they could not give or receive hugs as visiting was restricted by a glass between the detainees and the visitors.
We spoke with many people that afternoon. Jose [not his real name], a high school student, traveled more than 500 miles to see his father. Maria [not her real name] traveled over 200 miles to see her ex brother in law because after all he was still family. A large family group came through the gate with two boys, probably ages 8 and 9, crying over seeing their uncle. We simply stood outside, standing in solidarity with those detained and their families. A few of the sisters were able to provide information about local resources to assist families from outside of the area. At least one of our sisters has received clearance to visit the detention center; she also helps with translation.


Following the photography session, you may wish to join our dinner event to make new friends with the same interest, and to build your photography network. Dinner will be at Crazy Wings Chatswood (西门烤翅).
Set Menu includes: crazy wings, crazy lamb, crazy fish, fry rice/noodle, salard + drinks. Vegetarian menu also available (must RSVP in advance). Cost at $28 for Members and $35 for Non-Members. Create your Free Member Account Now!
To help us better organise the dinner event, please RSVP for part B by Thursday, 20th, January.
Click here to view "HOW TO FIND THE LIGHT HOUSE (MARK B)"
SYDPhoto is providing opportunities and support to its members to become one of the best photographers in Sydney.
SYDPhoto is looking for talented models to build their portforilos.
Most importantly, SYDPhoto would like its members to meet new friends and have fun!
Please go to our website to register as a photographer, model, stylist or member.
Then please send your nickname, profile ID and contact phone number to info@sydphotos.com.au. OR reply on the Photographers & Models Board this event topic.
For non-members, please register on Facebook or email your full name to info@sydphotos.com.au.
Part A: Arrive at the meeting location (google map above) on time to receive information about the event.
Citywalker magazine will publish the best photographer and model of this "CITYWALKER & SYDPHOTOS 2011 New Year Photography" event on Feb 2011.


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Home Hiking Destinations 6 Great Georgia Hiking & Backpacking Trails
6 Great Georgia Hiking & Backpacking Trails
Georgia hiking is blessed with wide terrain diversity from deep gorges and canyons to mountain summits, shoals to waterfalls, urban overlooks to historic sites, and rushing trout streams and wide rivers.
1 Georgia Hiking
1.1 Blood Mountain
1.2 Raven Cliff Falls
1.3 Kennesaw Mountain National Battlefield Park
1.4 Sweetwater Creek
1.5 Panther Creek
1.6 Amicalola River
Georgia Hiking
Here are six of the scenic hiking destinations to explore in the state of Georgia:
Blood Mountain
The Blood Mountain Trail is a looping style of path at 7.1 miles in length and situated near Blairsville, GA. A great feature of this trail is the scenic forest view.
It has an elevation gain of 1461-ft with heavy usability and rated as difficult, so a preferred course for the more experienced hiker.
The blood Mountain trail is open to the public from May to October and dogs are permitted provided a leash is used.
Raven Cliff Falls
The Raven Cliff Falls trail at 4.9 miles in length (there and back) is situated near Helen, GA and provides a stunning hike to a waterfall.
Other great features include its scenery, river, and forest. It has an elevation gain of 856-ft with heavy foot-traffic and regularly used for hiking, fishing, and camping. This is a dog-friendly Georgia hiking trail, but the dogs must be kept on a leash at all times.
Plus, the Raven Cliff Falls path is open year-round for hikers and backpackers to enjoy.
The Kennesaw Mountain National Battlefield Park is a loop trail at 7.25 miles in length and situated in Mariette, GA.
Great features of this trail include its wildlife, wildflowers, views, and forest. It has an elevation gain of 2660-ft with a medium difficulty rating and accessible to the public year-round. The main function for this path is trail running, hiking, and bird watching.
For easier access to the site of the civil war cannons, a shuttle bus can be taken to reach the 2 mile marker. Dogs can be taken on the trail provided a leash is used.
Sweetwater Creek
The Sweetwater Creek Trail is a looping path that is approx 4.3 miles in length and situated near Lithia Springs, GA.
This trail is relatively easy to navigate and open for hikers of all skill levels. It has an elevation gain of 332-ft and open to the public from September to May. Some of the most attractive features at Sweetwater Creek include its wildlife, waterfall, river, forest, and cave.
Usage in the region is low-medium and trail running and hiking is the most popular use. Dogs are allowed on the trail provided a leash is used.
The Panther Creek Trail is an out and back path at 7.1 miles in length and situated near Clarkesville, GA. It has an elevation gain of 740-ft with medium usage and popular for camping, walking, and hiking.
Plus, dogs are permitted on leash. Great features of Panther Creek include the waterfall, views, river, and forest.
Panther Creek is accessible to the public year-round with many changes in the scenery throughout the different seasons.
Amicalola River
The Amicalola River Trail is a loop style of path at 2.95 miles in length and situated near Dawsonville, GA.
It has light usage, a 450-ft elevation gain, and accessible to the public year-round. Great features of this moderate rated path include its scenery and rivers.
The primary function of the Amicalola River trail is walking and hiking.
Georgia hiking trail
hiking in georgia
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text:
Q: JWT::ExpiredSignature raised when validating token from getUserIdentityTokenAsync Our system is authenticating users by following these validation steps on a token generated by the getUserIdentityTokenAsync() method.
These tokens are failing the following check

Current time is between the times specified in the nbf and exp claims. The nbf claim specifies the earliest time that the token is considered valid, and the exp claim specifies the expiration time for the token. It is recommended to allow for some variation in clock settings between servers.

Our logs show these tokens being invalid by several hours
Current Time: 2020-04-10 17:02:11 +0000,
Valid Start: 2020-04-09 14:46:31 +0000,
Valid End: 2020-04-09 23:16:31 +0000,

We are validating these times as such:
    def timeframe_valid?
      PAYLOAD_WIGGLE_TIME = 900
      if @payload.present?
        now = Time.now.to_i
        payload_valid_start = @payload[:nbf] - PAYLOAD_VALID_WIGGLE_TIME
        payload_valid_expire = @payload[:exp] + PAYLOAD_VALID_WIGGLE_TIME

        if now < payload_valid_start || now > payload_valid_expire
          # handle invalid token...
        end
      end
    end

We are making a unique call to getUserIdentityTokenAsync before each api request. Since we are  seeing such a large difference in the time window for a valid token and the time it is validated, my best guess is that users are leaving the add-in open for long periods of time without refreshing. For some reason, when they return, getUserIdentityTokenAsync is not returning a new token, but rather the old one that is already expired.
Is there any way to force getUserIdentityToken to issue a new token after one has long since expired without a hard refresh of the add-in?

redpajama_set_name: RedPajamaStackExchange


Catholic blogger and novelist Kari Burke (author of the wonderful pro-life novel The Life I Dreamed) recently posted a review of Erin's Ring on her blog, From My Catholic Perspective.
Thanks so much, Kari! I am humbled and touched by your kind words, and so glad you feel that it's a book you can enjoy along with your daughters!


text:
« on: Wednesday, 29 December, 2021 @ 03:48:27 »
Thirty two new cases of coronavirus on Kef yesterday (in case you haven't seen yesterday's post)
Wed 29/12 16°/12° 13 km/h 73% Light rain probable
Sat 01/01 17°/11° 11 km/h 08% Sunny
Sun 02/01 17°/11° 7 km/h 10% Sunny
Thu 06/01 16°/11° 16 km/h 43% Showers possible
Fri 07/01 14°/10° 18 km/h 31% Showers possible
« Reply #1 on: Wednesday, 29 December, 2021 @ 08:08:32 »
Queen's Platinum Jubilee: Royal Mint releases new coins
A set of new coin designs have been released by the Royal Mint to mark the new year and the upcoming Queen's Platinum Jubilee.
The designs take years to develop and aim to mark memorable moments in history.
The £2 coins will have either a portrait of Dame Vera Lynn or a telephone - the invention of Alexander Graham Bell.
The 50 pence coin features a design to commemorate the Commonwealth Games, which will be held in Birmingham in 2022.
https://www.bbc.co.uk/news/av/uk-wales-59772837
https://youtu.be/gFNJSC6Pdmg?t=1
Magnitude 5.7 earthquake strikes Crete, felt in Egyptian cities
There were no immediate reports of injuries or damage.
https://www.reuters.com/business/environment/magnitude-61-earthquake-strikes-crete-greece-emsc-2021-12-29/
Magnitude Mw 5.7
Region CRETE, GREECE
Date time 2021-12-29 05:08:09.9 UTC
Location 34.85 N ; 25.09 E
Depth 68 km
Distances 53 km S of Irákleion, Greece / pop: 137,000 / local time: 07:08:09.9 2021-12-29
21 km S of Asímion, Greece / pop: 1,200 / local time: 07:08:09.9 2021-12-29
https://www.emsc-csem.org/Earthquake/earthquake.php?id=1082115
Greek divers discover Italian World War II submarine wreck
The Jantina, which had sailed from the Greek island of Leros with 48 sailors on board, sank on July 5, 1941, after being hit by torpedoes fired by British submarine HMS Torbay.
She was discovered last month by Kostas Thoctarides, one of Greece's best-known divers, and his team, south of the island of Mykonos at a depth of 103 meters using a remotely operated underwater vehicle.
https://www.ekathimerini.com/culture/1174656/greek-divers-discover-italian-world-war-two-submarine-wreck/
Minimum salary to rise 2+3%
The minimum salary is set to grow by 2%, from 650 to 663 euros per month, as of January 1, but with the prospect of a second increase within 2022, probably in the summer.
The benefit will be greatest for salary workers who have been in their position for at least nine years, as their minimum salary will rise from €845 to €861.90, with similar gains for recipients of other allowances, such as married employees.
https://www.ekathimerini.com/economy/1174562/minimum-salary-to-rise-2-3/
Hm, sounds like there's a promise from the Regional Council to have the road down to Myrtos and the 'Winter Bridge' fully restored in 2022. Hmmm.
Kef's wineries have requested that the amount of alcohol permitted in Robola be increased from a maximum of 12.5 to 13.5%. Apparently global warming and better techniques have increased the alcohol content in the wine with the result that the wine has had to have small amounts of wine from other grape varieties added to decrease the natural alcohol content. Currently, Robola with an alcohol content over the present 12.5% limit has been sold off as table wine.
Unusual weather led to 2021 being a bad year for the grapes, Robola production was down to around 200 tons, normally 600-700 tons are produced annually. Between 30-50% is exported.
Boris Johnson's Strange Explanation About Where He Spent Christmas Is Raising Suspicion
"I've been...I've been...in this country. Where do you think? Thank you."
While prime ministers are entitled to take holidays, Johnson's absence has raised eyebrows in recent weeks as Covid cases have been soaring across the UK.
He appeared to withdraw from public appearances as criticism of him increased in the wake of 'Partygate', while the ongoing allegations that No.10 held several parties for its employees in December 2020 infuriated the public, especially as the rest of the country was under strict lockdown rules at the time.
https://www.huffingtonpost.co.uk/entry/boris-johnson-absence-christmas-country-covid_uk_61cc4f80e4b061afe3a32d08
Amazon Alexa tells 10 year-old child to electrocute themselves for a 'challenge'
Amazon says that it has now removed the challenge from its database
https://www.independent.co.uk/life-style/gadgets-and-tech/amazon-alexa-penny-plug-challenge-b1983701.html
That Alexa, she's a bright spark
Rare albino puma cub discovered and rescued in Colombia
A rare albino puma cub has been discovered and rescued in Colombia, with vets now working to build up its strength for an eventual release back to a conservation park.
https://www.independent.co.uk/tv/climate/albino-puma-cub-rescued-colombia-v0fee27a7
https://youtu.be/GA1aqrCHesA?t=1
« Reply #10 on: Wednesday, 29 December, 2021 @ 18:58:46 »
Not sure how this has been produced, looks like a flight sim to me but I've not used one so can't say:
https://youtu.be/IBwx1BanaNo?t=1
Recent video of snow on Kef:
https://youtu.be/jRHnYpoiIC4?t=1
Ah, now this one has to be a flight sim, a Thomas Cook Airbus A321: Descent to Kefalonia Airport
Forty five new cases of coronavirus confirmed on Kefalonia today by EODY. Two cases on Ithaka.
« Last Edit: Wednesday, 29 December, 2021 @ 19:31:24 by Maik »
The Minister of Shipping and Island Policy has approved the construction of a metal water tank in the Faros area of Argostoli upto a cost of 250,000 euros.
Flooding: Ten alerts issued across East of England
Ten flood alerts have been issued across the East of England.
The Environment Agency issued the alerts, which means flooding is possible, for areas in Bedfordshire, Buckinghamshire, Cambridgeshire, Essex, Norfolk and Northamptonshire.
Due to "persistent rainfall", river levels across the counties are rising, the government agency said.
It has advised people to plan driving routes to avoid low lying roads near rivers.
The agency also urged people not to attempt to drive through flood water.
https://www.bbc.co.uk/news/uk-england-northamptonshire-59818191


"Non-asbestos Friction Materials" by Subramaniam Narayan, Brijnaresh R. Sinha et al.
The mechanical properties of composites made of six different non-asbestos fibers (mineral, phosphate, wollastonite, wollastokup, glass and carbon) and two resins (phenol-formaldehyde and cashew-modified phenolic) have been studied, in order to determine the binary interactions between the fiber and resin. The Young's modulus, ultimate tensile strength and flexural strength have been determined. The Young's modulus values have been compared with a theoretical model designed for random short fiber composites based on the Halpin-Tsai model with an empirical coefficient of 1/10. Scanning electron micrographs (SEM) of the fiber and fractured composite surface have been taken in order to determine the failure mode of the composites.
S. Narayan et al., "Non-asbestos Friction Materials," S.A.M.P.E. Quarterly, Society for the Advancement of Material and Process Engineering (SAMPE), Jan 1990.
© 1990 Society for the Advancement of Material and Process Engineering (SAMPE), All rights reserved.


text:
Guitar Practice Interview With Paul Nelson
Mike Philippov: Hi, this is Mike Philippov from PracticeGuitarNow.com. Today, I'm live on the phone with Paul Nelson, a very well-known and respected guitarist. with a very rare skill of playing exceptionally well in a broad range of styles. Paul has a lot of experience, both as a recording artist and a performer, as well as being a guitar instructor. So, I look forward to hearing his insights about practicing guitar. Welcome Paul and thank you for doing this interview.
Paul Nelson: Hi Mike. It's a pleasure to be here on the phone with you.
MP: My first question is how do you practice guitar to develop the confidence for playing well during live performances? How do you deal with nerves before the show?
PN: I, like a lot of other guitarists that I've read, that have been posed this type of question, really don't have an answer for that, but I understand the necessity of why students would want to know "Is there a time when you were feeling like I did" (meaning the students). It starts to come natural. At your first show or battle of the bands, or your first performance, you get a little bit of the jitters before you go on, but in my case, once I did it I was hooked. Nowadays, when working with Johnny Winter, he says he never gets it and I never get it either. You don't think - you do. All the practicing you have done ahead of time, all the exercises, all the chordal work, all the sight reading, all the schooling, all the teaching just comes out and that's the confidence. The confidence that you have done the work ahead of time, so nothing really sways you. When you have a lack confidence you realize you didn't do the work to prepare for whatever the situation is. It happens a lot in a sight reading situation, where charts are thrown in front of you, or you are in way over your head because you are just not ready for those type of situations, or that caliber of musicians. But, if you keep at it, it will happen for you.
MP: Which guitar playing skills did you have the most trouble developing and what practicing approaches did you use to finally master those skills?
PN: Any problem I had, I realized would start off with not being familiar with a certain musical style. In grooming myself as a session guy, I knew I had to play everything. I knew I had to wear different hats for different situations. If music changed, I knew I had to follow suit or innovate. What I did was when I started playing, if I felt I was weak in blues, or weak in metal, or weak in jazz or weak in rock or funk, I would form a band, and find musicians that played that one style and just delve into that musical style. So if it was funk, I started a James Brown/ Meters band. If it was blues - a Stevie Ray Vaughan band, and so on. We would go as far as going to a studio, a lot of people I know own home studios, and we would actually try to recreate the exact sound, and exact ideas for our favorite songs in the studio, and play it back like we had recorded that album too. Through that your own style comes. The familiarity of each style is what helps you along rather than going in there cold. I've got a lot more (session) work out of that, than a lot of other musicians who just play one certain style. That's the kind of musician I was bred to be - not just a one-style guy, which is very limiting. Nowadays you are credited for being versatile. You get to play with more people. You enjoy yourself more. You can make a career out of it, so it's important.
MP: How do you approach the process of warming up your hands before practicing or playing guitar whether it's just practicing at home or before a show? Do you have a regular warm up routine?
PN: In the beginning when I first started playing guitar, there were spider exercises where you run chromatically up and down the neck by one string at a time or you cross strings and you work on your alternate picking. I used to do those like crazy when I first started off and that was just to be able to play the notes. Then, years later, I actually met Steve Vai who gave me a ton of specific picking exercises, stretching exercises for each individual set of fingers, 1st and 2nd, and 2nd and 3rd and those types of exercises. Two fret per string exercises back and forth, working up the guitar neck and back down the neck horizontally, three sets per finger. And the big thing I noticed, after doing all those exercises was that if the ear wasn't there to hear those notes that you wanted to play, it hindered your speed and accuracy. The fingers can't play what the ears don't hear. Otherwise it's just guesswork. It really did require knowledge of ear training which I got at Berklee College Of Music, and I got from studying with Steve Kahn, and a bunch of other guys. It really helped me match the speed and accuracy with the ear, and then it all just clicks and starts happening. When I warm up, I listen to music that I like. I'll listen to some Freddie King, or I'll listen to Allan Holdsworth or Jeff Beck. I know a lot of drummers do that, before they go on to play they will listen to Neil Peart or Steve Gadd just to get inspired. It also helps you get out of a rut. Maybe you won't be able to play like that, but it locks you in the groove. Also a lot of metronome playing when you're home.
MP: What were the biggest sources of your guitar and musical education when you were developing your musical skills? You mentioned studying with Steve Vai and going to Berklee, but who were your other guitar teachers or places where you learned from?.
PN: I am a big fan of private instruction. I love Berklee. It was the type of school that when you were in it, you wanted to get out, but when you weren't in it you wanted to go back. Everybody was practicing 14 hours a day. But, I am also a huge fan of private guitar lessons. If you can afford it, find the best guys. I went straight to Manhattan and I found Steve Kahn and Mike Stern. Like I said, I found Vai in Boston. Steve Kahn was a huge influence. Music theory opened everything up for me how everything works. Then sight reading - Berklee was great for sight reading, and chords, and roman numeral analysis of song forms and chords, singing modes and hearing modes, that kind of thing. It was also great for meeting other musicians. We had 400 guitar players in one class at one time.
MP: In the process of learning to play guitar did you have moments of self-doubt when you compared yourself to your favorite guitar players and wondered if you were ever going to reach their level of guitar playing? If you had those feelings, how did you get past them?
PN: I think everybody has self doubt and that is what pushes you to want to get better. Blues guitarists in the past used to cut heads in these smoke-filled clubs. Buddy Guy used to battle BB (King) or whatever. I think it's the gentleman's sense of competition that makes you excel. I don't know if you ever know how good you've become. It's hard for a musician to hear himself. It's kind of like a guitar player, trying to actually listen to music. They always start tearing into the guitar parts, they always start looking for the little nuances. You're kind of doomed. It's hard to kick back and enjoy a tune. I do that - I enjoy it the first time, but when I hear it again, I am already starting to tear it apart: what's he doing and this and that. To know that you excelled, or get out of rut you have to have remain positive through constant practice, metronome work, scale work, and just a great knowledge of your craft. You have to know everything about it and then there's no room for messing up.
The thing about guitar players being geniuses to begin with and that guitar playing was already built into them - no, EVERYBODY has to practice. Even Robert Johnson, who "sold his soul to the devil", if you read his book, he sold his soul to the devil for 2 years, he's stunk, then 2 years later he got better. You have got to practice and that builds the confidence, then it happens. But, you can waste your time with bad guitar teachers, and waste your time with practicing the wrong stuff and learning the wrong guitar tab book, or going up the wrong musical path. The biggest thing is you how to get to where you want to get as quickly as possible and as accurately as possible. You have to really bone up and become a music historian on the stuff you want to play and listen to who influenced the guitar players that you like. You have to go back to the past.
MP: How do you go about identifying and correcting bad habits in your guitar techniques as you practice?
PN: It all comes through music theory, I believe. Most people say "I play with feel". Feel is great, but feel is crap if you don't know how to play stuff. You can't pick up a guitar, without ever playing it before in your life and say "Boy, do I have a lot of feeling", and then pick it up and play guitar like a monster. There's no way. How much of that is feel and how much of that is actual practice? Music theory allows you to analyze your (musical) development, find what mistakes you made, and know exactly where to go back to fix it. It's like fixing a car. If you hear a noise or a ping or something, a "feel guy" will get rid of the whole engine. But a theory guy will say "it's the carburetor or it's a hole in the exhaust and I'll fix that". It's the same thing with music theory. Music theory guys know if their rhythm is off or their knowledge of chords is off, or if they don't have a good ear for a certain mode or scale or phrase. Music theory really helps and I can't believe how much it really scares people. It probably scares them when you get into harmonic minor and melodic minor – then people just go bananas. Altered scales and all that kind of stuff, leading into a dominant approach stuff – that gets scary. But the modes and all that - you have to know that stuff. Study Mclaughlin, study Holdsworth.
MP: Speaking specifically about guitar technique, how do you practice guitar so that you can play consistently clean and accurately at faster tempos?
PN: There are 3 levels of guitar practice. One is called "practice-practice". And that means, you take scales and exercises and you work them with a metronome, just the way that the little instruction manual that comes with the metronome says how to work it. You work it like a robot. No feel, no emotion, you just "click, click, click, click, click, click, click". The second guitar practice phase is called "playing practice". Once you have mastered your scales and you have run them in intervals of 3rds and 4ths and 6ths and 7th and 5th now you want to play over chord changes. Play over a blues progression, play over a I vi ii V, play over a ii V I, play over modal changes. Now you are loosening up a little bit, but you are still playing over something purposely. So then you get ready for guitar practice part 3, which is playing with people. This is where the soul and the emotion comes in, and you play by feel. But soul and emotion and feel don't come into play until you have all of that militant guitar practicing under you and that's what allows you to add the soul and the feel. You can't play a riff you learned off a CD, with soul and feel, until you know it very well, then you play it with soul and feel, and put your own interpretation to it.
MP: I noticed a lot of guitar players get stuck in either the 1st or 2nd or 3rd method of practicing guitar you described. Few practice all three together, which is important like you point out.
PN: Yeah, bingo. Because after practicing all these picking exercises, and the long extended modal positions, and the alternate picking, the efficiency picking, the arpeggios, if you can't put it in a song and play over changes - you can't play! You can sit there and impress yourself, but you can't play with other people. If you don't know how to play over a major, minor or dominant - the three main families of music, you are screwed. But there are other guys who actually practice arpeggiating through a series of changes, which is good. You have to have chords playing in the background, or some kind of beat box, or some kind of rhythm going, so that it feels more like a natural guitar playing situation. Otherwise, if you just play freestyle, or practice freestyle, there's no point of reference to keep it in the groove when you play with a drummer.
MP: What is the process that you go through to practice memorization of songs or guitar solos? How do you practice so you don't forget your parts on stage, or when you play guitar in the studio?
PN: It's constant repetition. It could be cramming the night before. It could be cramming an hour before or a week before. There have been situations when I had to learn 40 songs, for one show, a week away because the guitar player backed out, or hasn't gotten a visa to cross over the country. So I've gotten the call: "can you do it?" So I've developed a shorthand system to write out song forms, to cue me for certain shapes. So it's repetition and that's the only way I memorize. The more you repeat, repeat, repeat, the more you will have it ready for the gig. However, when you cram like that, you have to be careful or it won't stick if you only play the songs once at a gig. If you get the gig and then you play those songs more and more, then it becomes a part of you. A lot of people learn something and then go to something else, then go to something else and you don't retain anything. You've got to be careful of that too, because you want to have it stick. The answer is that it is repetition, period. Everybody knows that's what you got to do. That's why you have guitarists sitting in a room for hours, and hours, and hours, and in their studio over and over again, driving everybody nuts.
MP: When you first started out playing guitar what goals did you have for yourself when you first began playing? How did those goals evolve for you over time?
PN: My goals, like any other guitar player, were to sound like the cool guy on the CD. That sound, that roughness, that when you listen to it you get a chill up your spine. I used to think, "Man, if I could play like that…", and it was just like a drug, depending on who you were listening to at the time. You were looking for guitar players who could get into your head and psych you into playing like that, then it just drove you. Listening to Randy Rhoads, or Eddie (Van Halen), Allan Holdsworth, or Jeff Beck or, Michael Schenker, or Yngwie or Stevie Ray Vaughn. Each guy does that. That's how you know that a guitar player is great, because he gives you that feeling. You strive to be like that. When you get that down you move to somebody else. The best guys are the combination of many artists, as you know. Vaughn was Albert King and Hendrix, Hendrix was Muddy, and Buddy Guy, and T-Bone. Even Johnny Winter, he is like a million guitar players because he listened to so many different guys.
MP: Do you believe there is a particular guitar playing skill that somebody must always practice and improve on a regular basis, regardless of how good they become as a guitar player?
PN: Picking is very important. Whatever style of picking you do, it's important. The best thing I have found is always going and buying CDs from the past. They are a little slower and a little easier to pick up. Always put some blues into your playing, so your stuff doesn't sound too stale. The blues, I think, is what actually puts emotion into your playing. As wild as Yngwie is, and those type of guys, the minute they would do whatever they did, and switch for a second into a cool bluesy run, you would say "that's cool". Eddie Van Halen, all the hundred great guitar players in Rolling Stone, all those guys had great blues-based ideas. And you really always have to go back and listen to Muddy, and a little Walter, Chuck Berry (if you can handle it). A lot of this stuff is really old and hard to listen to, but if you want to learn slide (guitar) for example, you have to listen to Muddy and Robert Johnson. Not just listen for a couple of seconds and then say, "Okay, I listed to Johnson, now I know how to do it". You really have got to delve into that and make it a part of you, to get something out of it. That's what all the guitar players who are great did. On a regular basis, yes, go back. It's okay to learn stuff from the guitar players who are current, but listen to the guys they listen to and that will put that extra juice into your guitar playing.
Discover the best way to solve your guitar playing problems - learn how to practice guitar to get HUGE results.


text:
\section{Introduction}

The broad range of applications for Green Fluorescent Proteins (GFPs)\cite{ grynkiewicz_new_1985,shaner_improved_2004-1,shimomura_extraction_1962} keeps them in the spotlight of researchers for many years now. However, it seems that once you think you have achieved almost perfect fluorescent proteins\cite{okabe_green_1997,lelimousin_intrinsic_2009}, new applications arise spanning a completely new field in fluorescent proteins research\cite{fernandez-suarez_fluorescent_2008, dedecker_fluorescent_2013,subach_chromophore_2012,kent_deconstructing_2008}. One such breakthrough was the discovery of the Reversibly-Switchable Fluorescent Proteins (RSFPs)\cite{habuchi_photo-induced_2006,habuchi_cover:_2005,hofmann_breaking_2005}. They are an indispensable tool in super-resolution microscopy\cite{dedecker_subdiffraction_2007, vandenberg_diffraction-unlimited_2015} and improvements to their photophysical properties are still being carried on \cite{bourgeois_reversible_2012,acharya_photoinduced_2016}. Theoretical studies have contributed significantly to understanding of RSFPs fluorescence quantum yield\cite{lelimousin_intrinsic_2009}, modulated isomerization quantum yield\cite{groenhof_photoactivation_2004} and color-tuning\cite{shcherbakova_red_2012}.

In spite of the large amount of research in this field, it seems like we still do not have a complete answer to the question why some proteins are photo-switchable while others are just fluorescent. How can only a few mutations in the protein $\beta$-barrel shift the absorption maxima by 40~nm\cite{andresen_photoswitchable_2008, faraji_nature_2015}? What is the role of the protein environment in this process and most importantly how does each aminoacid contribute to the photophysical properties of a certain protein?

There are a number of studies conducted on various fluorescent proteins identifying the role of protein environment on absorption maxima\cite{amat_spectral_2013} as well as emission and various electrostatic effects\cite{hasegawa_excited_2007, park_emission_2016}. Study on a set of green, orange and red FPs by Hasegawat et al.\cite{hasegawa_excited_2007} identified the blue-shifting effect of the protein electrostatic potential. Another detailed study on DsRed by List et al.\cite{list_molecular-level_2012} identifies the role of protein environment in one- and two-photon absorption. Also theoretical models were built to explain the pressure and temperature effects \cite{jacchetti_temperature_2016} or electrostatic field spectral tuning in GFPs\cite{drobizhev_long-_2015}. Studies on fluorescence and potential energy landscape of anionic GFP chromophore by Martin et al\cite{martin_origin_2004}  and Polyakov et al\cite{polyakov_potential_2010} provide in-depth mechanistic details for possible chromophore isomerization pathways. But in order to have an outlook which would allow for a rational protein design, a study on a consistent set of proteins is indispensable. That is why we have chosen a set of proteins differing by a minimal number of mutations, while conserving the chromophore structure. Step-by-step exploration of an excited state surface allowed us to connect absorption/emission, photo-switching speed and conical intersections with protein environment effects.

A well-known RSFP Dronpa\cite{habuchi_photo-induced_2006,moeyaert_green--red_2014} which was a pioneer in RSFPs field and is still widely used for various applications\cite{dedecker_fluorescent_2013,zhou_photoswitchable_2013}, became constitutive of the model protein set. Also, due to its pioneering role many Dronpa-like mutants were developed and well characterized\cite{andresen_photoswitchable_2008}. For our study we have selected Dronpa mutants which do not alter the chromophore structure, so all of the mutations occur in the $\beta$-sheets and/or $\alpha$-helix. The list of five proteins that were used in our study in shown in the Table \ref{tab:mutations}.

\begin{minipage}{\linewidth}
\centering
  \begin{tabular}{lllll}
    \hline
    Protein&Mutation&Absorption&Emission\\
    &&nm/kcal*mol\textsuperscript{-1}&nm/kcal*mol\textsuperscript{-1}\\
    \hline
    Dronpa\cite{moeyaert_green--red_2014}&&503/56.7&522/54.7\\
    rsFastLime\cite{stiel_1.8_2007}&V157G&496/57.5&518/55.1\\
    rsKame\cite{rosenbloom_optimized_2014}&V157L&503/56.7&515/55.4\\
    Padron0.9\cite{andresen_structural_2007, brakemann_molecular_2010}&T59M+V60A+N94I+P141L&504/56.6&510/56.0\\
    &G155S+V157G+M159Y+F190S&&\\
    bsDronpa\cite{andresen_photoswitchable_2008}&A69T+D112N+G155S+V157G&460/62.0&504/56.6\\
    &M159C+F173C&&\\
    \hline
  \end{tabular}\par
\captionof{table}{Effect of mutations on the absorption and emission wavelength in Dronpa-like mutants. Only the most essential mutations are indicated for Padron0.9. For bsDronpa six essential mutations are shown, which also were used to model it on the basis of Dronpa crystal structure.}
 \label{tab:mutations}
  \bigskip
\end{minipage}

Two single-point mutants rsKame\cite{rosenbloom_optimized_2014} and rsFastLime\cite{stiel_1.8_2007, andresen_photoswitchable_2008} were chosen due to their minimal difference with Dronpa. They do have a big difference in the speed of photo-switching (see Table S1), but there is barely any difference in the absorption and emission maxima. For a broader spectral variation we have chosen Padron0.9\cite{brakemann_molecular_2010} and bsDronpa\cite{andresen_photoswitchable_2008}. Both proteins conserve the chromophore structure with only a few mutations in the protein matrix. Padron0.9 is a negatively-switchable counterpart of Dronpa. But more importantly for us its absorption and emission maxima are slightly red-shifted in comparison to Dronpa. And bsDronpa is the most blue-shifted close mutant of Dronpa with a large Stokes shift (43~nm). Previous studies on FPs with large Stokes shift\cite{faraji_nature_2015, piatkevich_extended_2013, yoon_far-red_2016} have shown that the extended Stokes shift is a result of a more flexible hydrogen bond (H-bond) interplay where more states can be accessed in the ground and/or excited state surface. However, here our focus is on obtaining one bsDronpa population capable of reproducing the spectral trend of our model set. Hence we do not indulge into in depth conformational analysis of bsDronpa H-bonds fluctuations.

We would like to emphasize that the proteins used in our calculations differ by a maximum of 8 mutations and stem from the same protein thus minimizing the differences in the environment. Also, they contain exactly the same chromophore including its sidechain. Although the chromophore's $\pi$-system includes only phenol and imidazoline rings, the chromophore sidechain also plays an important role influencing the $\alpha$-helix flexibility and adaptability of the chromophore to the protein environment\cite{arpino_crystal_2012, moeyaert_green--red_2014, smyrnova_thermal_2016}.  Since we are interested in extrapolation of our results into functional mutations, conserving the protein structure is of a an extreme importance. The more mutations are included in the protein the less predictable become the results with increasing possibility of a failed experiment due to factors which are out of reach for computational studies.

Previous studies have shown how choice of an initial set up of the system can influence the calculated excitation energies\cite{filippi_bathochromic_2012,melaccio_towards_2016,amat_spectral_2013}. Here we have used exactly the same protocol for all five proteins, which can be briefly described as a modified Automatic Rhodopsin Modelling(ARM) protocol developed by Melaccio et al\cite{melaccio_towards_2016}. Starting QM/MM structures were taken from an extensive MD simulations. Then after CASSCF QM/MM optimization  CASPT2  method was used to calculated excitation and emission energies for a set of ten structures. Representative structures for each protein were chosen for further reaction pathway and charge transfer analysis. Charge transfer in the chromophore during excitation and emission is analyzed in correlation with the conformational changes in protein environment. Also, through calculation of individual aminoacid contributions to absorption/emission, we provide an explanation for a remarkable blue-shift in bsDronpa absorption spectra. And finally conical intersection and a closest lying minimum was identified for each of the proteins. As a result an excited state reaction pathway is assessed in a rational and unifying manner.

\newpage
\section{Results}
\subsection{Reproducing the experimental absorption and emission values}

\begin{figure}[H]
\includegraphics[width=13.5cm]{figures/Absorption_emissions_absorption.png}
  \caption{Comparison of the calculated (violet) and experimental (cyan) absorption and emission values for the model-set of five proteins. Results for the respective chromophores in vacuum are shown in orange. For the calculated values error bars are shown as well.}
  \label{fgr:trend}
\end{figure}

Results for the average absorption and emission values obtained in our study are shown in the Figure \ref{fgr:trend}. First of all, we would like to note that the absorption and emission trend is completely reproduced in our results. Not only a shift absorption/emission values is rather small, but also the order of proteins is conserved. Standard deviation of the average emission values with respect to the experimental emission values is around 1.5 kcal/mol (for all proteins). As it can be seen from the Figure \ref{fgr:trend} calculated absorption values are more blue-shifted than emission values (origins of that phenomena are discussed further in our paper). It is reflected in the standard deviation of the absorption average from the experimental values - from 2.66 kcal/mol (Padron0.9) to 4.63 kcal/mol (bsDronpa).

It can be noticed that mutations introduced in Dronpa (see Table \ref{tab:mutations}) can significantly affect the absorption maxima, while emission maxima are much less spread. There are two main sources affecting the absorption and emission maxima: the chromophore conformation and the protein environment. Since the chromophore structure is conserved in all five proteins we see the major source for absorption distribution in the way protein environment interacts with the chromophore in the ground and excited state. To discard the influence of the chromophore conformation we have calculated the absorption and emission values for the chromophore in vacuum (orange points in the Figure \ref{fgr:trend}). Since the trend is drawn on the average value over ten structures for each protein, we have used the most representative (closest to the mean values) conformation. As expected neither trend nor significant spread in the values can be found in these results. Hence it can be concluded that we should focus on the protein environment effect on the absorption and emission.

\subsection{Charge re-distribution in the chromophore. Origins of the higher absorption sensitivity to the mutations}
One of the most intriguing features of our model set is a 6 kcal/mol absorption maxima difference between Padron0.9 -- bsDronpa and  only 2 kcal/mol difference in emission between Dronpa -- bsDronpa.  Not only absorption is much more sensitive to mutations, but also there is no correlation with the emission. In another words, a higher absorption wavelength does not imply a higher emission maximum.

We have hypothesized that the electrostatic effects may play a pivotal role in this phenomena. Hence we have looked into the charge redistribution during the excitation and emission. Then the same calculation was repeated for chromophore $\pi$-system, but already in vacuum. The values presented in the Figure \ref{fgr:charge_redistribution} correspond to the charge redistribution in protein environment during the absorption and emission. The charge redistribution is essentially an atomic charge difference between the first excited and ground state.

\begin{figure}[H]
\includegraphics[width=15.5cm]{figures/charge_redistribution.png}
  \caption{Comparison of the charge redistribution in the chromophore during absorption and emission. The images are color coded with the color bar shown on the right. Values are indicated next to the atoms for which the charge difference exceeds 0.04. Hydrogen charges are summed into the heavy atoms.}
  \label{fgr:charge_redistribution}
\end{figure}

Blue and red colors in the Figure \ref{fgr:charge_redistribution} correspond to a positive and a negative charge differences. It can be noticed that the negative charge is transferred from the the aromatic phenol ring to imidazoline ring and the bridging atoms of the single-double bond bridge. This observation is in line with Filippi et al.\cite{filippi_bathochromic_2012}  results on anionic GFP. In the ground state $\tau$-bond has a prevalent double character while $\phi$ is a single bond (benzenoid resonance structure). Observed charge redistribution corresponds to a double-bond delocalization over $\tau$ and $\phi$ thus facilitating the isomerization (or shift to a quinoid resonance structure).

Although, charge redistribution pattern is similar during the absorption and emission the net charge transfer from phenol to imidazoline is more pronounced during the absorption (phenol ring is more blue and imidazoline ring is more red). When compared to the charge re-distribution in chromophore in vacuum no such difference was observed (see Figure~S1). Hence higher magnitude of the values spread in the absorption and not in the emission comes purely from protein environment.  First of all, mutations in the protein have direct effect on electrostatic potential around the chromophore. Also, pure spatial arrangement of the aminoacids around the chromophore can have important effects. In case when only one aliphatic aminoacid is mutated into another aliphatic one (Val 157 $\rightarrow$ Gly 157) minimal perturbation in electrostatic field around the chromophore is expected. However, slight rearrangement of the aminoacids around the chromophore can bring polar aminoacids closer to the chromophore and either red or blue-shift its absorption (individual influence of the aminoacids on spectral maxima is discussed further).

Charge transfer also results in a slight conformational change. The geometrical parameters that change the most in the proteins are depicted in the Figure~S2. Change in all of the parameters indicates that the chromophore is preparing for an isomerization. The negative charge is transferred from the phenol ring into the imidazoline ring and particularly the bridging carbon atom. This leads to phenol ring -- H-bonds weakening. Also the H-bonds between Arg~89, Arg~64 and imidazoline ring get stronger. Thus leading to a slight Glu~144 -- His~193 H-bond elongation and a consequent Glu~144 -- His~193 bond shortening. Finally, the double bond bridge is elongated indicating transfer to a single bond character. As a consequence there is a noticeable out-of-plane twisting around $\tau$ dihedral angle. It has been shown earlier by Morozov et al.\cite{morozov_hydrogen_2016} that a lower number of H-bonds with the chromophore induces the isomerization. A link between this observation and a cascade of hydrogen-bond-network (HBN) transformations triggered after the excitation is shown in our study.

\subsection{Protein environment influence on the absorption and emission maxima}

In order to see influence of the individual aminoacids on the absorption and emission maxima, we have performed calculations where their charges were consecutively turned off.  Results of the calculation can be see in the Figure S3.

The importance of His~193 due to it's $\pi$-stacking interaction with the phenol ring of the chromophore. H193T mutation as reported by Li et al\cite{li_primary_2010} shows how absence of this interaction induces the internal  conversion and leads to fluorescence loss. Interaction of the chromophore with Tyr~116 is often overlooked and our calculations clearly demonstrate its importance. To our knowledge there is no experimental data published for Dronpa Y116 mutants, however Y116Q and Y116N mutations in pcDronpa (green-to-red photoconvertible mutant of Dronpa)\cite{moeyaert_green--red_2014} lead to an increased photo-conversion quantum yield and higher pKa. The blue-shifting effect of Arg~89 clearly manifests itself in bsDronpa, where it loses interaction with the chromophore. Although, for a definitive answer an X-ray structure of bsDronpa must be awaited for we anticipate that a large Stokes shift occurs due to Arg~89 flexibility. When its interaction with the chromophore is switched off both in theory (for Dronpa -- rsKame -- rsFastLime -- Padron) and in practice (bsDronpa) a blue-shift in absorption is observed. The importance of Arg~64 and Arg~89 can be attributed to their strong interaction with the chromophore through HBN. Both Arg~64 and Arg~89 for H-bonds with imidazoline ring of the chromophore. When absent, the negative charge transfer is impeded thus increasing the energy of the excited state. Consecutively a blue shift occurs.

\subsection{Conical intersections on the isomerization path}

Once we had a working protocol to study photophysical properties of the fluorescent proteins we decided to go further and connect their photochemical reactivity with the properties of conical intersections.

Conical intersections (CIs) very likely act as bottlenecks for the isomerization reactions in photo-activatable proteins. In particular, properties of the CI region should be crucial for photoswitching speed of the RSFPs. However in order to prove this an assessment of a group of photoswitching proteins is needed. At the same time there is an inverse correlation between the speed of photoswitching and fluorescence quantum yield (as seen in the Table S1). That suggests a pivotal role of excited state reaction kinetics due to possible presence of exited state energy barriers as well as distinctive topography of the conical intersection.

To identify conical intersections on the excited state isomerization path of RSFPs we have used a conical intersection optimization with a constrain module available in Molcas\cite{fdez._galv?n_analytical_2016}. CI optimizations were carried out at CASSCF(12,11)/6-31G* level and afterwards a single-point SA-CASPT2(12,11)/6-31G* calculation was done on optimized structures.

Results of our findings can be seen in the Figure~\ref{fgr:CI} and relative energies are summarized in the Table~S2. For the triad rsFastLime -- Dronpa -- rsKame the conical intersection energies are inversely proportional to their isomerization quantum yields (and the photoswitching speed). Namely, rsFastLime is the fastest switcher of all three and respectively it's CI has the lowest energy (60.2~kcal/mol), then follows Dronpa (61.6~kcal/mol) and rsKame -- the slowest switcher -- has the highest CI energy of all three (66.7~kcal/mol). On the basis of the results for these three proteins we could have concluded that the energy of CI guides the photoswitching speed. Following this conclusion we would have expected the CI energy of Padron0.9 and bsDronpa be even lower than rsFastLime energy, since both of them exhibit extremely fast switching behavior. However, following numerous attempts on CI optimization, it was not possible to lower the CI energies of Padron0.9 and bsDronpa and they have stayed at 78.3kcal/mol and 76.3 kcal/mol respectively. Hence, not only kinetics but also topology of the CI region is important for photoswitching.

\begin{figure}[H]
\begin{subfigure}[b]{0.55\textwidth}
\includegraphics[width=9.1cm]{figures/mechanism_Dronpa.png}
\end{subfigure}
\begin{subfigure}[b]{0.40\textwidth}
\includegraphics[width=8.5cm]{figures/mechanism_Padron.png}
\end{subfigure}
  \caption{Potential energy surface of the chromophore isomerization. Black lines correspond to the mechanism proposed for rsKame -- Dronpa --rsFastLime and blue lines are for bsDronpa -- Padron. The energy values are indicated in the corresponding order and color.  Dashed and continuous lines correspond to the ground and excited state surfaces respectively. TS region is indicated in grey in it's hypothetical position.}
  \label{fgr:CI}
\end{figure}

To characterize the CI region we have also identified a local minimum which would be the closest to the CI point. So, for each minimization the structure identified as a CI point was a starting structure and minimization proceeded with no constraints.
For rsFastLime -- Dronpa -- rsKame an energy minimum was found in a vicinity of a CI point with energies of 54.4, 55.9 and 65.1 kcal/mol respectively. Energy minima for Padron0.9 and bsDronpa were identified at 60.6 and 56.5 kcal/mol respectively. However, after visual inspection and comparison of the ground state energies it was established that the minimized structures belong to Franck-Condon region. In order to quantify the extent of the isomerization reaction coordinate we have used calculated the RMSD of the chromophore from the initial minimum ground state structure. RMSD comparison can be found in the Table~S2 and superimposed chromophore structures from FC and CI regions are shown in the Figure S4.  Compiling all of the findings it can be deduced that the CI region of rsFastLime -- Dronpa --rsKame is separated by an energy barrier from a Franck-Condon region, while in case of Padron0.9 and bsDronpa it can be accessed directly. Our results are in accordance with the potential energy surface described by Polyakov et al.\cite{polyakov_potential_2010} for a HBDI chromophore. Authors indicate a possibility of a higher-lying CI with and a low excited-state transition state. At the same time they identify a low-lying CI and a high energy transition state. As such the fast switching of bsDronpa and Padron0.9 can be explained by a flat potential energy surface which directly connects FC and CI regions.

\section{Conclusion}

By selecting a consistent set of reversibly-switchable fluorescent proteins (Dronpa -- rsFastLime -- rsKame -- Padron -- bsDronpa) we have built a protocol completely reproducing their absorption and emission trend. With only a slight blue-shift of only about 2 kcal/mol it provides a solid tool for rational fluorescent protein design and a mechanistic study.

A thorough investigation of an excited-state reaction path allowed us to rationalize photoswitching properties. Via charge transfer analysis and individual aminoacid contributions it has been shown that absorption is more sensitive to mutations and changes in the protein environment than emission. A general cause of this phenomena we see in higher susceptibility of the excited state species. Sometimes denominated as "hot" species \cite{vengris_contrasting_2004}, which are unequilibrated with the protein environment. During the excitation the system is in equilibrium in the ground and not excited state. While during the emission system founds itself in equilibrium in the excited state. Hence environmental changes disturb the system more during the absorption and less during the emission. bsDronpa with its remarkable Stokes shift serves as an example of the protein environment flexibility. The most representative population was identified during the MD simulations. The major difference from the rest of the proteins is n absence of a hydrogen bond between Arg~89 and the imidazoline ring of the chromophore. Our individual aminoacid calculations clearly show Arg~89 contributing to a blue shift in absorption, which is reproduced in bsDronpa.
Further on, an excited state reaction pathway is assessed for a set of photoswitching proteins. It is shown how conical intersection controls the speed of photoswitching as well as brightness. However, it has to be noted that an existence of an excited state energy barrier also play an important role in the protein's photophysics. In general, a higher lying conical intersection is separated by an excited state energy barrier from Franck-Condon region. However, in case of the fastest switching proteins (Padron and bsDronpa) no such barrier was identified. And although conical intersection in these two proteins has a higher energy than in the slower switching ones, it is the flat potential energy surface which facilitates the isomerization. A more accessible conical intersection relates to a lower brightness and a higher isomerization speed.

Further characterization of the conical intersection topology, such as it's slopeness, as well as excited state classical trajectory calculations could provide more mechanistic details.

\section{Computational Methods}

\textit{Molecular Dynamics (MD) and Replica-Exchange MD (REMD)}

As the first step vast conformational sampling of all five proteins has been performed using Gromacs 5.0.1\cite{pronk_gromacs_2013} package and the Amber99sb-ILDN forcefield\cite{case_amber_2014}. In case of Dronpa and Padron0.9 X-ray structures of the bright states were used (2Z10\cite{mizuno_light-dependent_2008} and 3ZUJ\cite{regis_faro_low-temperature_2011} respectively). rsKame, rsFastLime and bsDronpa were modelled by point mutations on the basis of Dronpa crystal structure. The list of mutations differentiating Dronpa from the rest of the proteins is given in the Table \ref{tab:mutations}.

In detail description of MD simulation for Dronpa, rsFastLime and rsKame can be found in our previous work\cite{smyrnova_molecular_2015, smyrnova_thermal_2016}. A similar scheme has been adopted for Padron0.9 and bsDronpa. PROPKA \cite{li_very_2005} predictions were used to determine protonation states of the titratable residues. In case of Padron0.9 we have adopted the protonation scheme proposed by Brakemann et al.\cite{brakemann_molecular_2010}, where GLU 144 is protonated and HIS 193 is neutral with a proton placed on N$\epsilon$. Crystal waters present in the X-ray structure were conserved. Then proteins were solved in a 80.5$\times$80.5$\times$80.5 {\AA}$^3$ TIP3P water box and neutralized by Na\textsuperscript{+} and Cl\textsuperscript{-} counterions added at the concentration of 0.15M. Prepared systems were minimized and equilibrated at 300K temperature and 1bar pressure for 100ps. Long-range interactions were included using Particle Mesh Ewald method, cut-off distance was set 13.5\AA. Since a good quality crystal structure was available for Padron0.9, five 100~ns MD runs. were enough for sufficient conformational sampling. A time step of 2~fs and SHAKE algorithm\cite{ryckaert_numerical_1977} were employed. The temperature was controlled using velocity rescaling with a stochastic term\cite{bussi_canonical_2007}.
bsDronpa model has been constructed by introduction of 6 point-mutations (listed in the Table 1) to Dronpa structure. In order to assure a thorough conformational search a Temperature Replica-Exchange MD (T-REMD) simulation was performed. Twenty structures were assigned temperature values from 300K to 330 K which were obtained using the temperature generator server for REMD-simulations\cite{patriksson_temperature_2008}. Equilibration was performed at each of 20 temperatures following the same procedure as described for MD simulation. Then 50~ns runs were performed for each replica thus leading to 1$\mu$s cumulative production run. The resulting exchange probability was $\approx$ 20$\%$ with exchange attempts occurring every 2~ps.

GROMACS utilities were used to perform cluster analysis for each of five proteins. As a results the median structure (the most representative for each simulation) was determined. Then these structures were used as input structures for QM/MM simulations.
\newline
\textit{The QM/MM calculations}

MOLCAS/Tinker interface\cite{aquilante_molcas_2010} was used to perform QM/MM calculations. MOLCAS 7.8\cite{aquilante_molcas_2010} was used to describe QM part and Tinker 5.1\cite{ponder_efficient_1987} for MM part. In preparation of the QM/MM models the Automated Rhodopsin Modeling (ARM)\cite{melaccio_towards_2016} approach was used. This tool has proved to be a robust protocol for simulations of rhodopsin-like photoreceptors. Certainly there is structural difference between Dronpa-like fluorescent proteins and rhodopsin-like photoreceptors. However similarity in their photobiological characteristics allows to employ the same approach.

The QM/MM model was constructed on the basis of a median structure obtained from classical MD simulations. Proteins were re-centered in the water box, then a sphere of 30{\AA} radius from the chromophore was defined. Since no periodic boundary conditions are employed during the QM/MM simulations this approach allows to save computing time without losing the environment effect. QM/MM model can be divided into three parts: environment, cavity and QM part. Schematic representation of the QM/MM models constructed for five proteins is shown in the Figure~S5.

The input structure used for QM/MM model was subject to 10 $\times$1~ns MD, where the environment was kept frozen and cavity atoms flexible. Then 10 random frames were picked up from the resulting trajectories and used for consecutive QM/MM calculations. Thus for each protein 10 independent QM/MM models were prepared.

\begin{figure}[H]
\includegraphics[width=13.5cm]{figures/Dronpa_sphere_chrom.png}
  \caption{Full system is shown on the left (protein shown as cartoon, Na+  and Cl- ions as spheres (blue and violet respectively), dark cyan surface indicates a 30{\AA} water sphere and chromophore is shown as sticks). A 4{\AA} radius cavity around the chromophore was defined and comprised of X residues (cyan) including the chromophore (green). All this residues were flexible during the optimization. The water molecules present in the crystal structure were maintained and included in the cavity. Chromophore was included in the QM region.}
  \label{fgr:7}
\end{figure}

All the atoms corresponding to the environment are fixed. The 4{\AA} radius cavity was defined around and including the chromophore. Residues in the cavity were free to relax. The QM part comprised of the chromophore's $\pi$-system with link atoms\cite{singh_combined_1986} placed between carbon atoms (shown in the Fig. X). Link atom position was restrained according to Morokuma scheme\cite{svensson_oniom:_1996}. The charge of the frontier atoms was set to 0 and the residual fractional charge redistributed of the rest of chromophore atoms according to atomic mass and insuring -1 integer charge of the chromophore. In all calculations MM atoms were described with Amber94\cite{cornell_second_1995} force field.

As the first step structures were optimized at HF/3-21G/MM level. Then a single-point CASSCF(12,11)/6-31G*/MM calculation was performed in order to define orbitals, which correspond to $\pi$-orbitals and excluding the ones with population close to 0 or 2. Orbitals defined in single-point calculation were used as input for the consecutive CASSCF(12,11)/6-31G*/MM optimization. Then we switch to a CASPT2(12,11)/6-31G*/MM 3-single root single point calculation is performed, taking a 3-roots State Average (SA)\cite{ferre_approximate_2002} CASSCF(12,11)/6-31G*/MM wavefunction as reference. As a result 10 vertical excitation energies ($\Delta$E\textsubscript{S1-S0}) were computed. Then CASSCF(12,11)/6-31G*/MM excited-state optimization was performed. For the located excited-state minimum structure a single-point CASPT2(12,11)/6-31G*/MM 3-single root calculation was performed. Emission energies were computed as the difference between the first two roots ($\Delta$E$'$\textsubscript{S1-S0}). Average over 10 values for each protein was taken as a reference for comparison to the experimental values.

To calculate the standard deviation from the experimental values we have used the standard formula given in Eq.\ref{eqn:STD}
\begin{equation}
  \sigma = \sqrt{\frac{1}{N} \sum_{i=1}^N (x_i - x_{exp})^2}\label{eqn:STD}
\end{equation}
where N=10 and x\textsubscript{exp} is an experimental value.

Charge re-distribution shown in the Figure \ref{fgr:charge_redistribution}  was calculated as a difference between the Mulliken charges in the ground and excited states. Hydrogen charges were summed up to the heavy atoms.

To calculate the effect of each aminoacid on absorption and emission maxima the most representative structures for each protein were used for single-point CASPT2(12,11)/6-31G*/MM 3-single root calculation. Charges of atoms corresponding to each amino acid were set 0 in Tinker key-file.

\begin{acknowledgement}
Financial support from the Flemish Government through the KU Leuven concerted action scheme is gratefully acknowledged.
The computational resources and services used in this work were provided by the VSC (Flemish  Supercomputer Center), funded by the Hercules Foundation and the Flemish Government.

\end{acknowledgement}

\begin{suppinfo}
Following information can be found in the Supporting Information:
computational details,.

\end{suppinfo}

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